EDSON DA SILVA
EFEITOS DO DIABETES EXPERIMENTAL SOBRE A MORFOLOGIA E
FUNÇÃO DO VENTRICULO ESQUERDO DE RATOS WISTAR PÚBERES:
IMPACTO DO TREINAMENTO DE NATAÇÃO
Tese apresentada à Universidade
Federal de Viçosa, como parte das
exigências do Programa de PósGraduação em Biologia Celular e
Estrutural, para obtenção do título de
Doctor Scientiae.
VIÇOSA
MINAS GERAIS – BRASIL
2013
EDSON DA SILVA
EFEITOS DO DIABETES EXPERIMENTAL SOBRE A MORFOLOGIA E
FUNÇÃO DO VENTRICULO ESQUERDO DE RATOS WISTAR PÚBERES:
IMPACTO DO TREINAMENTO DE NATAÇÃO
Tese apresentada à Universidade Federal de
Viçosa, como parte das exigências do
Programa de Pós-Graduação em Biologia
Celular e Estrutural, para obtenção do título
de Doctor Scientiae.
APROVADA: 08 de novembro de 2013.
_____________________________
Luciana Duarte Novais Silva
______________________________
Ita de Oliveira Silva
______________________________
Sirlene Souza Rodrigues Sartori
_______________________________
Silvia Almeida Cardoso
________________________________________
Izabel Regina dos Santos Costa Maldonado
(Orientadora)
i
O saber a gente aprende com os mestres e os livros.
A sabedoria se aprende é com a vida e com os humildes.
Cora Coralina
ii
Dedico à minha esposa Marileila,
e aos que me dedicaram suas vidas:
meus pais, Luíza e Gedi (presente na memória),
e aos meus irmãos Noêmia, Luís e Eduardo.
iii
AGRADECIMENTOS
A Deus pela vida e pela coragem de continuar a caminhada em todos os momentos.
À Marileila, minha esposa, amiga, companheira de todos os momentos, pelo exemplo
de vida e pela força em superar tantas dificuldades e caminhar hoje ao meu lado.
À minha mãe Luíza, meus amados irmãos Noêmia, Luís, Eduardo e seus familiares,
ao Salvador e sua família, por tudo que sempre fizeram por mim e por tudo que cada
um representa ao longo da minha trajetória até aqui. Agradeço a todos pelo amor
incondicional e pela representação de um modelo de esperança e fé.
Aos amigos do Laboratório de Biologia Estrutural: Ana Cláudia, Mariana, Carol,
Suelem, Kyvia, Juliana, Kener, Marli, Wagner, Marta, Tatiana, Graziela, Suzana,
Daiane, Camila, Stéphanie, Bruno e Bárbara, pela disposição em ajudar, pelas
conversas e momentos de descontração, pela companhia na luta do dia-a-dia e pelo
incentivo constante.
Aos companheiros de trabalho Mateus, Alex Bhering, Eliziária e Daise por
iluminarem nossos caminhos em meio aos procedimentos e técnicas para as análises,
pelo apoio nos experimentos, pela paciência, companhia e os adoráveis momentos de
descontração.
Aos amigos do coração que fazem o dia-a-dia mais feliz: Claúdia, Eliziária, Kener,
Rômulo, Gilton e Mírian.
À minha orientadora Profª. Izabel Regina dos Santos Costa Maldonado, por me
acolher novamente na UFV. Obrigado pela parceria, confiança, paciência, amizade,
pelos ensinamentos e apoio em questões acadêmicas, pessoais e profissionais. Uma
pessoa exemplar e uma professora admirável.
Ao meu coorientador Prof. Antônio José Natali, pela orientação, disposição em
ajudar, pelo apoio com o projeto junto à FAPEMIG, e incentivo para realizar os
trabalhos e por ceder à infraestrutura do seu laboratório para realização de
experimentos.
Ao meu coorientador Prof. Leandro Licursi de Oliveira, pelos conselhos em
pesquisa, pela solicitude em ajudar e abrir as portas do seu laboratório para a
realização de experimentos.
Aos professores do Laboratório de Biologia Estrutural, Adilson, Sérgio, Mariana,
Juliana, e Clóvis pela colaboração na análise do material histológico, no uso de
equipamentos e materiais e pelo exemplo de profissionalismo.
iv
Ao Departamento de Biologia Geral da UFV pela confiança em nosso trabalho de
equipe, pelo apoio financeiro para os experimentos e solicitude em ajudar.
Ao Prof. José Eduardo Serrão pelos estímulos constantes para a busca de qualidade
nas pesquisas, pelo apoio financeiro e de infraestrutura para a utilização dos
laboratórios da UFV e realização de algumas etapas de experimentos.
Aos professores Gustavo Ferreira Martins e Jorge Abdalla Dergan por gentilmente
permitirem a utilização de seus laboratórios para aquisição das imagens utilizadas.
À Universidade Federal de Viçosa e ao Programa de Pós-Graduação em Biologia
Celular e Estrutural, pelo apoio em minha formação profissional e pessoal.
A todos os professores do Programa de Pós-Graduação em Biologia Celular e
Estrutural, por todos os ensinamentos e incentivo.
Aos funcionários do Programa de Pós-Graduação em Biologia Celular e Estrutural,
em especial Beth e Diana, pela ótima convivência, paciência, amizade e apoio
sempre.
Às professoras Sirlene Souza Rodrigues Sartori, Ita de Oliveira Silva e Mariella
Bontempo Duca de Freitas pelas orientações no ensino em anatomia humana e
fisiologia. Pelo exemplo de simplicidade, profissionalismo, caráter e amizade.
Sempre aprendo com vocês.
Aos colegas do Biotério Experimental do Departamento de Educação Física pela
colaboração nos experimentos, apoio e convivência.
À Daise pela colaboração ao longo dos experimentos, pelos conselhos, dedicação,
paciência, alegria, pelo exemplo de profissionalismo e de caráter e pela amizade.
À Marcinha pela parceria na pesquisa, pelos ensinamentos com o modelo
experimental do diabetes, pelo exemplo de garra e disposição para o trabalho.
À FAPEMIG, pelo apoio financeiro.
À CAPES, pela concessão da bolsa de pesquisa durante parte do meu doutorado.
À Universidade Federal dos Vales do Jequitinhonha e Mucuri e aos colegas do
Departamento de Ciências Básicas, especialmente aos professores Amauri, Paulo
Messias e Luiz Gabriel por concederem o meu afastamento para a realização do meu
doutorado.
v
À Ieda Baracho dos Santos, funcionária do Departamento de Ciências Básicas da
UFVJM pelas inúmeras orientações, informações e solicitude em ajudar a qualquer
momento.
Às professoras Luciana Duarte Novaes Silva, Ita de Oliveira Silva, Sirlene Souza
Rodrigues Sartori e Silvia Almeida Cardoso pela disposição em participar da banca
examinadora.
vi
SUMÁRIO
LISTA DE ABREVIATURAS E SIGLAS...............................................................viii
RESUMO....................................................................................................................xii
ABSTRACT ............................................................................................................ xiii
INTRODUÇÃO GERAL .............................................................................................1
REFERÊNCIAS BIBLIOGRÁFICAS........................................................................ 6
ARTIGO 1..................................................................................................................13
ARTIGO 2 .................................................................................................................38
CONCLUSÕES GERAIS......................................................................................... 71
vii
LISTA DE ABREVIATURAS E SIGLAS
AMPK – Proteína quinase ativada por AMP
ANOVA – Análise de variância
ATP – Adenosina trifosfato
ATPase – Enzima que catalisa a hidrólise do ATP
BG – Blood glucose
bmp – Beats per minute
BW – Body weight
Ca2+ – Íon cálcio
CaCl2 – Calcium chloride
CaMKII – Calcium/calmodulin-dependent protein kinase II
CE – Controle exercitado
CEUA – Ethics Committee on Animal Experimentation
CRP – C-reactive protein
CS – Controle sedentário
DCM – Diabetic cardiomyophaty
DE – Diabético exercitado
dL – Decilitro
DM – Diabetes mellitus
DS – Diabético sedentário
EC – Exercise control
ECG – Electrocardiogram
ECM – Extracellular matrix
ED – Exercise diabetic
EGTA – Ethylene glycol-bis (ß-aminoethyl ether)-N, N, N’, N’-tetraacetic acid
ELISA – Enzyme linked immunosorbent assay
eNOs – Endothelial nitric oxide synthase
g – Gramas
GLUT 1 – Transportador de glicose 1
GLUT 4 – Transportador de glicose 4
Hepes – N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
HF – Heart failure
viii
HMW – High molecular weight
HR – Heart rate
Hz – Hertz
ICAM-1 – Intercellular adhesion molecule-1
IL – Interleukin
IUR – Uniform random section
KCl – Cloreto de potássio
Kg – Quilograma
L – Litro
LMW – Low molecular weight
LV – Left ventricle
M – Molar
mg – Miligrama
MgCl2 – Cloreto de magnésio
min – Minuto
mL – Mililitro
mM – Milimolar
MMW – Middle molecular weight
ms – Milissegundo
NaCl – Cloreto de sódio
NCX – Na/Ca Exchange
ng – Nanogramas
NO – Nitric oxide
ºC – Grau Celsius
PAS – Periodic acid-Schiff
pH – Potencial hidrogeniônico
PLB – Phospholamban
PN– Programa de natação
PPARγ – Receptor ativado por proliferadores de peroxissoma gama
RyR2 – Canais receptores de rianodina
S1P – Esfingosina-1 fosfato
SC – Sendentary control
SD – Sedentary diabetic
ix
SEM – Standard error of the mean
SERCA2 – Sarcoplasmic reticulum Ca 2+ ATPase
SphK1 – Esfingosina quinase 1
SR – Sarcoplasmic reticulum
STZ – Estreptozotocina
T1DM – Type 1 diabetes mellitus
T2DM – Type 2 diabetes mellitus
TNF-α – Fator de necrose tumoral alfa
VCAM-1 – Vascular cell adhesion molecule-1
VE – Ventrículo esquerdo
vs – Versus
VW – Weight gain
WG – Ventricular weight
% r.c.l – Percentage of resting cell length
µg – Microgramas
µm – Micrômetro
µM – Micromolar
μL – Microlitro
x
RESUMO
SILVA, Edson da, D.S., Universidade Federal de Viçosa, Novembro de 2013.
Efeitos do diabetes experimental sobre a morfologia e função do ventrículo
esquerdo de ratos Wistar púberes: impacto do treinamento de natação.
Orientadora: Izabel Regina dos Santos Costa Maldonado. Coorientadores: Antônio
José Natali e Leandro Licursi de Oliveira.
A cardiomiopatia diabética está associada não só com o remodelamento cardíaco e
disfunção miocárdica, mas também com a ocorrência de inflamação de baixo grau e
redução dos níveis de adiponectina cardíaca, resultando em significativa morbidade e
mortalidade em pacientes com Diabetes mellitus tipo 1 (DM1). Por outro lado, o
exercício físico é uma estratégia importante para o tratamento do diabetes, que pode
reduzir a inflamação, atenuar o remodelamento cardíaco adverso e a disfunção
contrátil. O objetivo deste estudo foi investigar a influência do treinamento de
natação de baixa intensidade no remodelamento estrutural, nos níveis de citocinas
cardíacas, na inflamação e na disfunção contrátil de cardiomiócitos do ventrículo
esquerdo (VE) de ratos púberes com diabetes experimental não tratado. Ratos Wistar
machos, com trinta dias de idade, foram divididos em quatro grupos (n = 19 por
grupo): controle sedentário (CS), controle exercitado (CE), diabético sedentário (DS)
e diabético exercitado (DE). O diabetes foi induzido por estreptozotocina (STZ, 60
mg kg-1 de peso corporal). Animais dos grupos CE e DE foram submetidos a um
treinamento de natação (5 dias/semana , 90 min/dia, com carga de 5 % do peso
corporal) durante 8 semanas. Após a eutanásia, o VE foi removido para análises
molecular, morfológica e mecânica de cardiomiócitos. Secções do VE foram coradas
com Periodic acid-Schiff (PAS), Sirius Red, reticulina de Gomori, tricrômico de
Gomori e azul de toluidina/borato de sódio 1%. O VE de animais diabéticos
apresentou aumento de colágeno intersticial e fibras reticulares na matriz
extracelular, acúmulo de glicogênio, desorganização da histoarquitetura do
miocárdio,
infiltrado
inflamatório
e
necrose.
A
densidade
capilar
foi
significativamente menor nos animais diabéticos. Cardiomiócitos do VE dos animais
diabéticos apresentaram o tempo para o pico de contração e o tempo para 50% de
relaxamento mais longos do que os animais do grupo controle, mas não houve
diferença na amplitude de contração. Os níveis cardíacos de IL-10, óxido nítrico,
adiponectina total e adiponectina HMW foram significativamente menores nos
xi
animais diabéticos. O exercício físico atenuou o nível de TNF-α e os parâmetros
histopatológicos avaliados, e aumentou a densidade capilar no VE de ratos
diabéticos. Em conclusão, o remodelamento estrutural cardíaco induzido pelo DM1
experimental coexiste com níveis reduzidos de adiponectina total e HMW,
inflamação crônica e disfunção de contratilidade dos cardiomiócitos. Mais
importante ainda, o treinamento de natação de baixa intensidade atenuou parte destas
alterações patológicas, indicando o papel benéfico do exercício regular no DM1 não
tratado.
xii
ABSTRACT
SILVA, Edson da, D.S., Universidade Federal de Viçosa, November, 2013. Effects
of experimental diabetes on the morphology and function of the left ventricle of
pubescent rats: impact of swimming training. Adviser: Izabel Regina dos Santos
Costa Maldonado. Co-advisers: Antônio José Natali e Leandro Licursi de Oliveira.
Diabetic cardiomyopathy is associated not only with cardiac remodeling and
myocardial dysfunction but also with the occurrence of low-grade inflammation and
reduced cardiac adiponectin resulting in significant morbidity and mortality in
patients with type 1 diabetes mellitus (T1DM). On the other hand, physical exercise
is an important strategy for the management of diabetes which can reduce
inflammation and attenuate adverse cardiac remodeling and contractile dysfunction.
The aim of this study was to investigate the influence of low-intensity swimming
training on the structural remodeling, cardiac cytokines, inflammation, and
cardiomyocyte contractile dysfunction of the left ventricle (LV) in pubescent rats
with unmanaged experimental diabetes. Thirty-day-old male Wistar rats were
divided into four groups (n = 19, per group): sedentary control (SC), exercised
control (EC), sedentary diabetic (SD), and exercised diabetic (ED). Diabetes was
induced by streptozotocin (STZ, 60 mg kg-1 body weight). Animals from EC and ED
groups were submitted to a swimming training (5 days/week, 90 min/day, load of 5%
body weight) for 8 weeks. After euthanasia LV was removed for molecular,
morphological, and cardiomyocyte mechanical analysis. Sections of LV were stained
with Periodic acid-Schiff (PAS), Sirius Red, Gomori’s reticulin, Gomori's trichrome,
and toluidine blue/sodium borate 1%. The LV of diabetic animals presented
increased interstitial collagen and reticular fibers on the extracellular matrix,
accumulation
of
glycogen,
myocardial
histoarchitectural
disorganization,
inflammatory infiltrate and necrosis. The capillary density was significantly lower in
diabetic animals. Left ventricular cardiomyocytes from diabetic animals exhibited
more prolonged time to the peak of contraction and the time to half relaxation than
those from control animals, but no difference in cell shortening was observed. The
cardiac levels of interleukin 10, nitric oxide, total and HMW adiponectin were
significantly decreased in diabetic animals. Exercise training attenuated the level of
TNF-α and the histopathological parameters assessed, and increased the capillary
density in the LV of diabetic rats. In conclusion, the cardiac structural remodeling
xiii
induced by experimental T1DM coexists with reduced levels of total and HMW
adiponectin, chronic inflammation and cardiomyocyte contractility dysfunction.
More important, low-intensity swimming training attenuated part of these
pathological changes which indicate the beneficial role for regular exercise in
untreated T1DM.
xiv
INTRODUÇÃO GERAL
O Diabetes mellitus (DM) é uma desordem metabólica caracterizada pelo
aumento da glicemia resultante de defeitos na secreção de insulina, ação da insulina,
ou em ambos (AMERICAN DIABETES ASSOCIATION, 2013); com alta
prevalência em adultos e adolescentes (MA, CHAN, 2009). O número de crianças,
adolescentes e jovens adultos com DM aumenta a um ritmo alarmante (STEHNOBITTEL, 2012; TRUONG et al., 2012). As doenças cardiovasculares representam a
principal complicação do diabetes e podem ter início na infância (NADEAU et al.,
2011) e influenciar de forma significativa a morbidade e a mortalidade na fase adulta
(RAJESH et al., 2012; GIANNINI et al., 2011; GUO et al., 2007; LIBBY et al.,
2005) .
A
maioria
dos casos de diabetes divide-se em
duas categorias
etiopatogenéticas: Diabetes Mellitus tipo 1 (DM1) e tipo 2 (DM2) (AMERICAN
DIABETES ASSOCIATION, 2013). No DM1 a causa é uma deficiência absoluta da
secreção de insulina, enquanto no DM2 a causa é uma combinação da resistência à
ação da insulina e uma resposta secretora inadequada de insulina (AMERICAN
DIABETES ASSOCIATION, 2013).
O DM1 é uma doença que ocorre principalmente por deficiência de insulina,
a partir da destruição das células beta pancreáticas (AMERICAN DIABETES
ASSOCIATION, 2013) e as doenças cardiovasculares podem estar associadas à
resistência à insulina nesta forma de diabetes (PEREIRA et al., 2012; NADEAU et
al., 2010; KILPATRICK et al., 2007; DEFRONZO et al., 1982). Além disto, o DM1
tem sido relacionado à inflamação de baixo grau e às complicações micro e
macrovasculares em adultos (LLAURADÓ et al., 2012; GONZALEZ-CLEMENTE
et al., 2007) e crianças (MANGGE et al., 2004). A inflamação sistêmica de baixo
grau pode ser definida como a elevação, em duas a quatro vezes, das concentrações
de citocinas que desencadeiam ações pró e anti-inflamatórias (BRUUNSGAARD,
2005).
As vias que levam à disfunção cardíaca induzida por DM e suas alterações
histopatológicas ainda não estão totalmente esclarecidas (NADEAU et al., 2010;
SALEM et al., 2009; NEMOTO et al., 2006), em parte, devido à natureza complexa
e multifatorial do diabetes (LAW et al., 2012). Possivelmente, o papel mais
importante tem sido atribuído à hiperglicemia persistente (LI et al., 2012), condição a
1
qual aumenta os riscos para o desenvolvimento de insuficiência cardíaca de duas a
cinco vezes (BELL, 1995).
Pessoas com diabetes podem desenvolver uma disfunção cardíaca
denominada cardiomiopatia diabética (LAW et al., 2012). Esta é caracterizada por
dilatação e hipertrofia do miocárdio, acompanhada por disfunção sistólica e/ou
diastólica do ventrículo esquerdo (VE) e a sua presença é independente da
coexistência de doença cardíaca isquêmica ou hipertensão (HAYAT et al., 2004).
Apesar de a cardiomiopatia diabética ser uma condição subclínica crônica
(VOULGARI et al., 2010), eventualmente leva à redução da elasticidade da matriz
extracelular e diminuição da função contrátil do coração (FALCÃO-PIRES, LEITEMOREIRA, 2012; RAJESH et al., 2012; ARAGNO et al., 2008; SEARLS et al.,
2004). A associação entre o desenvolvimento da cardiomiopatia diabética e os
elevados níveis de glicose têm efeitos significativos sobre a expressão, organização e
modificação de componentes da matriz extracelular no coração (LAW et al., 2012).
A matriz extracelular cardíaca é uma rede dinâmica bem definida, constituída
de proteínas estruturais, proteoglicanos, fatores de crescimento, citocinas e enzimas,
essenciais para a organização e estrutura do tecido (LAW et al., 2012). É composta
por colágeno (CAULFIELD, BORG, 1979), sendo colágeno fibrilar dos tipos I e III
localizados no interstício do miocárdio, e colágeno não fibrilar dos tipos IV e VI e as
glicoproteínas fibronectina e laminina, predominantes na membrana basal dos
cardiomiócitos (BROWER et al., 2006; BISHOP, LAURENT, 1995; EGHBALI,
WEBER, 1990). Elevações no estresse do miocárdio iniciam um remodelamento
estrutural do coração, na tentativa de normalizar o estresse imposto. Este processo
compreende a hipertrofia de cardiomiócitos com mudanças na quantidade e no
fenótipo do colágeno, ocorrência de ligações cruzadas (cross-linking) entre
moléculas de colágeno e remodelamento progressivo de componentes musculares,
vasculares e da matriz extracelular do coração (BROWER et al., 2006). O aumento
das concentrações intersticiais de colágeno e/ou de ligações cruzadas resulta em
rigidez do miocárdio e disfunção diastólica do ventrículo esquerdo (BROWER et al.,
2006).
Adicionalmente, no DM a inflamação de baixo grau pode estar relacionada ao
aumento dos níveis sanguíneos de proteínas pró-inflamatórias, tais como fator de
necrose tumoral alfa (TNF-α), interleucina-6 (IL-6), proteína C-reactiva (PCR) e à
redução dos níveis de proteínas anti-inflamatórias (LLAURADÓ et al., 2012; RYBA
2
et al., 2011). Um estado inflamatório crônico ocasiona alterações histopatológicas
com lesão tecidual, disfunção endotelial e remodelamento cardíaco, os quais
resultam em deterioração da contratilidade miocárdica e da função ventricular
esquerda (LLAURADÓ ET AL., 2012; DUNCAN ET AL., 2007). A redução do
transiente de Ca2+ (DUNCAN et al., 2007) e a redução da sensibilidade dos
miofilamentos contrácteis ao Ca2+ são mecanismos possivelmente envolvidos neste
processo (GOLDHABER et al., 1996).
Estudos confirmam que a adiponectina desempenha um papel importante no
metabolismo de glicose e de lipídios (PEREIRA et al., 2012; GAREKANI et a.l,
2011; NUMAO et al., 2008), além de seu impacto relevante na patogênese do
diabetes, da resistência à insulina e da lesão vascular (FENGER, 2013; GU et al.,
2012). A adiponectina é uma proteína plasmática, secretada principalmente pelos
adipócitos (BOBBERT et al., 2011; SUN, CHEN, 2010) e possui propriedades
antidiabéticas, anti-inflamatórias, antiapoptóticas, antiaterogênicas (FORSBLOM et
al., 2011; BOBBERT et al., 2011), imunomoduladoras e cardioprotetoras (JENKE et
al., 2013; LETH et al., 2008). Dentre outras células, os cardiomiócitos são capazes de
sintetizar adiponectina (PIÑEIRO et al., 2005; MAIA-FERNANDES et al., 2008), a
qual tem função autócrina potencializando seu efeito cardioprotetor (HUI et al.,
2012). As vias de sinalização dos efeitos cardioprotetores da adiponectina são
mediadas principalmente pelo aumento de proteína quinase ativada por adenosina
monofosfato (AMPK), receptor ativado por proliferadores de peroxissoma gama
(PPARγ), esfingosina-1 fosfato (S1P) e esfingosina quinase 1 (SphK1) (HUI et al.,
2012).
Existem três principais isoformas de adiponectina: adiponectina de baixo
peso molecular (low molecular weight, LMW), médio peso molecular (middle
molecular weight, MMW) e alto peso molecular (high-molecular weight, HMW)
(HICKMAN, WHITEHEAD, 2012). Acredita-se que a adiponectina HMW é a
isoforma mais ativa nos tecidos periféricos (GOTO et al., 2013; MAIAFERNANDES et al., 2008). Há evidências de que a adiponectina protege o coração
de lesões isquêmicas, cardiomiopatias e disfunção sistólica (GOLDSTEIN, SCALIA,
MA, 2009). Além disto, a adiponectina é capaz de inibir a hipertrofia patológica dos
cardiomiócitos e a fibrose do miocárdio (MAIA-FERANDES et al., 2008; HAN et al,
2007), reduzir o stress oxidativo e nitrativo (WANG et al., 2013; GOLDSTEIN,
SCALIA, MA, 2009), inibir a expressão de TNF-α e de IL-6, e aumentar a expressão
3
de IL-10 no coração (LO, MITSNEFES, 2012; GOLDSTEIN, SCALIA, MA, 2009;
MAIA-FERANDES et al., 2008; HAN et al., 2007).
Por outro lado, o exercício físico regular é parte importante no manejo do
DM1, devido aos efeitos benéficos à saúde, especialmente a prevenção de doenças
cardiovasculares (GALASSET, RIDDELL, 2013). Assim, o treinamento físico tem
sido recomendado como estratégia não farmacológica útil para reduzir a resistência à
insulina, aumentar o metabolismo da glicose e de lipídios (KHAN, 2013), atenuar as
alterações morfológicas e a disfunção contrátil em animais e humanos com
cardiomiopatia diabética no DM1 (SILVA et al., 2013; CHIMEN et al, 2012;
LOGANATHAN et al., 2012; RAJESH et al, 2012; SILVA et al., 2011; BIDASEE et
al, 2008; SEARLS et al., 2004; DE ANGELIS et al., 2000). De modo geral,
melhorias na função contrátil do miocárdio são observadas em seres humanos e
animais, in vivo, em corações isolados e preparações multicelulares, submetidos a
diferentes modelos de exercício (DI BELLO et al., 1996).
Além disto, a atividade física regular está associada à longevidade e à menor
incidência de complicações decorrentes do diabetes (YARDLEY et al., 2012).
Enquanto o treinamento físico de alta intensidade pode promover remodelamento
cardíaco patológico em ratos (BENITO et al., 2011), o exercício aeróbico de baixa
intensidade melhora a aptidão física e a força, reduz fatores de risco cardiovascular,
melhora o bem-estar, reduz a necessidade de insulina e melhora a função autonômica
cardíaca (CHIMEN et al, 2012; YARDLEY et al., 2012; DE ANGELIS et al., 2000).
A literatura comprova que o exercício aeróbico de baixa a moderada intensidade é
capaz de induzir adaptações cardiovasculares benéficas (por exemplo, melhora a
função cardíaca, a estrutura do miocárdio, reduz a pressão arterial, e aumenta a
sensibilidade baro e quimiorreflexa, o ritmo cardíaco intrínseco e o débito cardíaco),
em ratos com diabetes induzido por estreptozotocina, um modelo que imita muito o
DM1 humano (JORGE et al., 2012; LOGANATHAN et al., 2012; MOSTARDA et
al., 2009; LOGANATHAN et al., 2007; DE ANGELIS et al., 2000). No entanto, os
mecanismos subjacentes à histopatologia do DM1 e sua relação com o exercício
físico, o remodelamento cardíaco e a disfunção cardíaca não são claros. Diante disto,
torna-se importante conhecer os ajustes estruturais e morfológicos do miocárdio após
o treinamento físico.
A regulação da glicose ou do metabolismo lipídico por adiponectina, em
resposta ao treinamento físico, têm sido investigada (GARENAKI et al., 2011;
4
ANDO et al., 2009) principalmente com análises dos níveis circulantes de
adiponectina total e HMW (ANDO et al., 2009). Até o momento, poucos estudos
examinaram a relação de fatores que influenciam os níveis de adiponectina em
crianças e adolescentes com DM1, e os resultados destes estudos são inconsistentes
(KARAMIFAR et al, 2013; MENON, 2012; HABEEB et al., 2011; MESSAAOUI,
2012; HUERTA, 2006). Estudos recentes exibem redução na expressão de
adiponectina cardíaca em ratos com diabetes induzido por estreptozotocina (PEI et
al, 2013), porém, a relação entre adiponectina cardíaca em ratos púberes com DM1 e
os efeitos de um programa de treinamento de natação nunca foi investigada.
Embora os relatos evidenciem de remodelamento cardíaco em humanos e
animais adultos, dados semelhantes em ratos púberes com DM1 são escassos
(SILVA et al., 2013; LAW et al., 2012; BROWER et al., 2006). Crianças e
adolescentes com diabetes têm muitos dos benefícios que os adultos adquirem com a
prática de exercícios, por isto devem praticar exercício físico com segurança, tanto
para a saúde como para o lazer (ROBERTSON et al., 2009). Uma vez que os ratos
são considerados um modelo animal válido para a compreensão do DM e são
constantemente utilizados para este fim, acreditamos que ratos jovens podem servir
como modelos para orientar o desenvolvimento de novas estratégias de prevenção e
tratamento do DM.
Deste modo, o presente estudo foi realizado para verificar a influência do
treinamento de natação de baixa intensidade sobre a estrutura dos cardiomiócitos, o
remodelamento estrutural do miocárdio, níveis de citocinas e de óxido nítrico, e a
densidade capilar do miocárdio, assim como a disfunção contrátil de cardiomiócitos
do VE de ratos púberes com diabetes experimental não tratado com insulina.
5
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12
Artigo 1
Ventricular remodeling in growing rats with experimental diabetes: the impact
of swimming training
Edson Silvaa,b, Antônio J. Natalic*, Márcia F. Silvaa, Gilton J. Gomesc, Daise N.Q.
Cunhac, Regiane M.S. Ramosc, Marileila M. Toledo d, Filipe R. Drummondc, Felipe
G. Belfortc, Rômulo D. Novaesa,e, Izabel R.S.C. Maldonado a*≠
*
a
The authors share senior authorship.
Department of General Biology, Federal University of Viçosa, Viçosa, MG, Brazil.
b
Department of Basic Sciences, Federal University of Jequitinhonha and Mucuri
Valleys, Diamantina, MG, Brazil.
c
Department of Physical Education, Federal University of Viçosa, Viçosa, MG,
Brazil.
d
Department of Medicine and Nursing, Federal University of Viçosa, Viçosa, MG,
Brazil.
e
Department of Biological Sciences and NUPEB, Federal University of Ouro Preto,
MG, Brazil.
Keywords: Fibrosis; ventricular remodeling; collagen matrix; diabetes mellitus;
exercise.
≠
Address for correspondence: Izabel Regina dos Santos Costa Maldonado,
Universidade Federal de Viçosa, Departamento de Biologia Geral. Edifício Chotaro
Shimoya, Avenida PH Rolfs, s/nº, CEP: 35.570-000 Viçosa, Minas Gerais, MG,
Brasil. Tel: +55 (031) 3899-3365. Email: [email protected]
Silva E, Natali A J, Silva M F, Gomes GJ, Cunha DN, Ramos RSN, Toledo MM,
Drummond FR, Belfort FG, Novaes RD, Maldonado, I. R. (2013). Ventricular
remodeling in growing rats with experimental diabetes: The impact of swimming
training. Pathology-Research
and
Practice, 209(10),
618-626.
DOI:
10.1016/j.prp.2013.06.009.
13
Abstract
Diabetic cardiomyopathy is associated with cardiac muscle remodeling, resulting in
myocardial
dysfunction,
whereas
exercise
training
(ET)
is
a
useful
nonpharmacological strategy for the therapy of cardiac diseases. This study tested the
effects of low intensity swimming training on the structural remodeling of the left
ventricle (LV) in growing rats with unmanaged experimental diabetes. Thirty day old
male Wistar rats were divided into four groups (n = 5/group): sedentary control (SC),
exercised control (EC), sedentary diabetic (SD), and exercised diabetic (ED).
Swimming training rats exercised 5 days/week, 90 min/day, with a load of 5% BW
during 8 weeks. Sections of LV were stained with Periodic acid Schiff, Sirius Red,
and Gomori’s reticulin. Seven days and 8 weeks after streptozotocin (STZ) induction
(60 mg kg-1 BW), blood glucose (BG) in the diabetic groups (SD = 581.40 ± 40.48;
ED = 558.00 ± 48.89) was greater (p < 0.05) than in their controls (SC = 88.80 ±
21.70; EC = 85.60 ± 11.55). Swimming-training reduced BG by 23 mg dL-1 in the
diabetics (p > 0.05). The LV of diabetic rats had increased interstitial collagen and
reticular fibers on the extracellular matrix and presented glycogen accumulation.
More importantly, all these adverse tissue changes induced by STZ were attenuated
by ET. Together, these findings support the idea of a beneficial role of exercise in the
LV remodeling in rats with unmanaged type 1 diabetes mellitus.
14
Introduction
Diabetes mellitus (DM) and its long-term complications are considered one of
the major health problems worldwide [33]. The number of children, adolescents, and
young adults with DM continue to rise at a dramatic rate [54,55]. In patients with
diabetes, cardiovascular complications represent the chief cause of morbidity and
mortality in adults [46], children, and adolescents [20].
The pathways leading to DM-induced cardiac dysfunction and its
histopathological changes are yet to be fully clarified [42,44,48]. However, this could
be in part due to the complex and multifactorial nature of diabetes [31]. Probably the
most important role has been attributed to persistent hyperglycemia [33]. This
condition increases the risks for developing heart failure (HF) from two- to five fold
[8].
Individuals with diabetes can develop cardiac dysfunction, termed diabetic
cardiomyopathy (DCM) [31]. Diabetic cardiomyopathy is characterized by
myocardial dilatation and hypertrophy, accompanied by systolic and diastolic left
ventricle (LV) dysfunction, and its presence is independent of the coexistence of
ischemic heart disease or hypertension [25]. Although DCM may be subclinical for a
long time [58], eventually, it leads to decreased myocardial elasticity and impaired
heart contractile function [3,18,46,49]. The link between elevated glucose levels and
the development of DCM has significant effects on the expression, organization, and
modification of extracellular matrix (ECM) components in the heart [31].
The cardiac ECM is a dynamic well-defined network consisting of structural
proteins, proteoglycans, growth factors/cytokines, and enzymes that provide essential
cues and scaffolding for tissue organization and structure [31]. It is composed of
collagen [14], with fibrillar collagen types I and III localized within the myocardial
interstitium and with nonfibrillar collagen types IV and VI, and the glycoproteins
fibronectin and laminin predominating in the cardiomyocyte basement membrane
[11,17]. Elevations in myocardial stress initiate structural remodeling of the heart in
an attempt to normalize the imposed stress. This process comprises cardiomyocyte
hypertrophy and changes in the amount of collagen, collagen phenotyping, and
collagen cross-linking with progressive remodeling of the muscular, vascular, and
ECM components of the heart [12]. Increased interstitial collagen concentrations
15
and/or cross-linking results in a stiffer myocardium and ventricular diastolic
dysfunction [12].
Exercise training is of particular interest in both the prevention and treatment
of DCM in type 1 DM [35]. Thus, exercise training has been suggested to be a useful
nonpharmacological strategy to attenuate morphological changes and contractile
dysfunction in both animals and human with DM [10,15,16,49]. Furthermore, regular
physical activity is associated with greater longevity and lower frequency and
severity of diabetes complications [59]. While long-term intensive exercise training
is suggested to promote adverse remodeling (i.e., fibrosis) in a rat model [9], lowintensity aerobic exercises improves physical fitness and strength, reduces
cardiovascular risk factors, improves well-being, reduces insulin requirements, and
improves cardiac autonomic function [15,16,59]. Indeed, low- to moderate-intensity
aerobic exercise has been shown to induce beneficial cardiovascular adaptations
(e.g., improves cardiac function, myocardial structure; reduces blood pressure;
increases baro- and chemoreflex sensitivity, intrinsic heart rate, and cardiac output)
in STZ-induced diabetic rats, a model that imitates many of the human type 1 DM
[16,29,34,35,41]. Nevertheless, the mechanisms underlying the histopathology of
type 1 DM and their relationship to physical exercise, myocardial interstitial fibrosis,
and cardiac functional impairment are unclear. Although reports show evidence of
cardiac remodeling in adult humans and animals, similar data in growing rats with
type 1 DM are lacking [12,31]. Children and adolescents with diabetes should have
many of the same health and leisure benefits as adults and should be allowed to
practice physical exercise with safety [47]. Since rats are considered a valid animal
model for understanding the DM and are consistently used for this purpose, we
believe that young rats could serve as models for guide the development of novel
strategies for prevention and management of DM. Therefore, the present study was
undertaken to verify the effects of low-intensity swimming training on the structural
remodeling of the LV of growing rats with unmanaged experimental diabetes.
Materials and Methods
Animals and experimental groups
Male Wistar rats weighing 87.40 ± 13.03 g, 30 days old, were obtained from
the animal facility at the Federal University of Viçosa, Brazil, and were randomly
16
divided into four groups (n = 5, per group): sedentary control (SC), exercised control
(EC), sedentary diabetic (SD), and exercised diabetic (ED). Rats were maintained on
12-h dark/light cycle at 22°C, housed in groups of five, and fed standard commercial
rodent chow and water ad libitum. All procedures followed the Guidelines for Ethical
Care of Experimental Animals and were approved by the Ethics Committee on
Animal Experimentation (CEUA) from the Federal University of Viçosa (protocol
number 46/2011).
Diabetes induction
Severe diabetes was induced in the animals by intraperitoneal injection of
streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO) dissolved in 0.1 M citrate buffer
solution (0.1 M, pH 4.5) at the dose of 60 mg kg-1 body weight (BW) [28].
Equivalent volume (1 mL kg-1) of vehicle was injected into the rats assigned to the
control groups. Animals were fasted overnight for 12 h prior to STZ administration.
Water and food were available immediately after dosing. Development of diabetes
was determined by observing hyperglycemia (> 300 mg dL-1) [51] as measured by an
Accu-Chek
Advantage
glucometer
(Boehringer
Mannheim
Corporation,
Indianapolis, IN). Body weights and blood glucose levels were recorded once a week
throughout the study. All animals were euthanatized 8 weeks after diabetes induction
by intraperitoneal injection of sodium pentobarbital (120 mg kg-1).
Exercise protocol
After seven days of diabetes induction and confirmation of consistent
hyperglycemia, animals from the exercised groups (ED and EC) were submitted to a
swimming training program (adapted from Gomes et al. [22]) for 8 weeks. Rats were
placed in water tank with 65 cm high by 75 cm in diameter, filled with warm water
(28°C to 30°C) at a depth of 45 cm and forced to swim. Animals were then dried
and returned to the home cage. Training intensity varied by changing a load that was
placed around the animal’s chest from 0% to 5% of its BW. Briefly, in the first week,
animals exercised in the water for 10 to 50 min, with no load, while duration was
increased by 10 min each/day. In the second week, animals remained exercising with
no load and with the duration incremented by 10 min/day until a maximum of 90 min
of continuous swimming. From the fourth week, animals began swimming with a
load until the end of the training program (8 weeks). The load was progressively
17
increased by 1% of the animal’s BW from the fourth week on, such that at the eighth
week, animals swam with a total load of 5% of their BW. During the swimming
sessions, animals from the sedentary group (SD and SC) were placed in a
polypropylene box containing warm water (28°C to 30°C) with a depth of 10 cm.
Electrocardiogram
Animals were anesthetized in an induction chamber with 2% isoflurane and
100% oxygen at a constant flow of 1 L/min. Once unconscious, they were placed on
a platform in dorsal recumbency, with the four limbs fixed. Isoflurane was
maintained at a concentration sufficient for restrain (0.5% to 1.0%), and animals
were able to maintain spontaneous respiration during the electrocardiogram (ECG).
A trichotomy of approximately 1 cm2 was performed in the forelimbs and left
hindlimb for electrode insertion. Derivation II of the ECG was recorded using the
data acquisition system PowerLab® (AD Instruments, São Paulo, Brazil), and data
analysis was done with the program LabChart Pro® (ADInstruments LabChart 7, São
Paulo, Brazil).
Electrocardiograms were performed at the end of the experiments by an
experienced fellow blind to the study groups and treatments. Resting heart rate (HR)
was derived from the ECG, following established guidelines [26].
Histological processing and histochemistry
Following euthanasia, the heart was excised, washed with saline solution, and
fixed by immersion in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH
7.2–7.4, for 24 h. The LV was dissected and weighed separately. Left ventricle
fragments were obtained through the orientator method to define isotropic and
uniform random sections (IUR) required in the stereological study [38]. These
fragments were dehydrated in ethanol, cleared in xylol, and embedded in paraffin.
Blocks were cut into 5 μm sections and mounted on histological slides. The LV
sections were stained by Periodic acid-Schiff (PAS) for glycogen, Sirius Red (for
total collagen), and Gomori’s reticulin (for silver impregnation of reticular fibers,
which mainly consist of collagen type III) to evaluate the series of histopathological
changes in the diabetic myocardium. Digital images were captured using a light
microscope (mod. Primo Star, Carl Zeiss AG, Oberkochen, Germany) connected to
a digital camera (AxioCam ERc5s, Carl Zeiss AG).
18
Analysis of myocardial collagen content
To quantify interstitial fibrosis, LV was evaluated in histological sections 5
µm thick stained with Sirius Red dye (Sirius Red F3B, Mobay Chemical Co., Union,
NJ, USA), which marks collagen fibers types I and III for further observation under a
polarizing microscope [30]. The distribution of total collagen content was analyzed
using the image analysis software Image Pro-Plus 4.5 ® (Media Cybernetics, Silver
Spring, MD, USA) based on the birefringence properties of the collagen fibrils under
polarized light. In this analysis, 12 microscopic fields from five rats per group were
investigated (magnification ×200) randomly. A 208-point grid was superimposed on
each image, and the total of 2,496 points was assigned for each rat. The area was
determined by adding up these points, then dividing the result by the total points for
the cross-section. Results were expressed as mean ± SD. Samples were observed
under an Olympus BX53 microscope equipped with a DP-73 digital camera and
imaging system (CellSens, Olympus Corporation, Tokyo, Japan).
Periodic acid-Schiff and Gomori’s reticulin staining
A qualitative analysis based on the intensity of the staining reaction with the
ventricular muscle was performed using PAS stain and Gomori’s reticulin stain. In
this analysis, 12 microscopic fields from five rats per group were investigated
(magnification ×1,000) randomly. Tissue sections of LV were stained with PAS for
detection of polysaccharides [53], and amylase-treated sections served as negative
controls. Other sections of LV were stained by Gomori’s reticulin. The level of
polysaccharides/collagen staining received a score that varied from 1 cross (+),
representing weak reaction for polysaccharides/collagen fibers, to 4 crosses (++++),
representing the strongest staining for polysaccharides/collagen (adapted from
Spillman et al. [53]).
Statistical analysis
Results were expressed as mean ± SD. The Kolmogorov-Smirnov test for
normality was initially performed. Statistical differences were evaluated by two-way
ANOVA or unpaired t-test when appropriate, and the post hoc Tukey test was
applied for multiple comparisons. The analysis was performed using the Sigma Stat
software, version 3.0, and statistical significance was defined as p ≤ 0.05.
19
Results
The animals of the groups SD and ED developed the expected hyperglycemia
of severe diabetes compared with control groups. Initially, on the baseline, there
were no differences in blood glucose levels between diabetic (SD vs. ED) and control
rats (SC vs. EC) (Table 1). Blood glucose increased throughout the experiment in
both SD and ED rats. Seven days after the application of STZ and at the end of the
study, the glycemic levels for the diabetic groups (SD and ED) were significantly
greater compared with their controls (Table 1). Swimming training reduced BG level
by 23 mg/dL in the diabetic groups, but this did not reach statistical difference.
The analyses of the baseline body weights were not different among the four
groups (Table 1). Diabetic rats had lower BW gain compared with normal rats (SC
and EC) (p < 0.05). Similarly, the LV weighted significantly less in the diabetic rats
(SD and ED).
Table 1. Biometrical and functional parameters
Parameters
SC (n = 5)
EC (n = 5)
SD (n = 5)
ED (n = 5)
Baseline BW, g
90.40 ± 3.29
93.60 ± 17.64
78.00 ± 17.78
87.60 ± 6.43
Final BW, g
353.60 ± 63.01
345.60 ± 63.56
174.80 ± 50.49 *
175.00 ± 43.89 †
WG, g
263.20 ± 63.03
252.00 ± 76.80
96.80 ± 40.68 *
87.40 ± 43.07 †
VW, mg
364.20 ± 40.90
354.60 ± 104.84
231.60 ± 43.28*
198.00 ± 47.94†
1.32 ± 0.15*
1.13 ± 0.30 †
VW/BW, mg/g
1.03 ± 0.27
1.03 ± 0.34
Baseline BG, mg/dL
66.20 ± 9.04
65.40 ± 5.50
73.40 ± 12.10
73.80 ± 11.13
BG Post-STZ mg/dL
72.40 ± 6.02
63.40 ± 9.02
418.80 ± 98.98 *
480.60 ± 68.34 †
Final BG, mg/dL
88.80 ± 21.70
85.60 ± 11.55
581.40± 40.48 *
558.00± 48.89 †
Resting HR, bmp
352.58 ± 17.07
300.50 ± 47.60*
256.6± 12.83 *
266.34± 31.29
Data are presented as mean ± SD; n, number of animals; SC, sedentary controls; EC,
exercised controls; SD, sedentary diabetics; ED, exercised diabetics; BG, blood
glucose; BW, body weight; VW, ventricular weight; WG, weight gain; HR, heart
rate. *, different from SC; †, different from EC; (p < 0.05).
20
As expected, STZ-induced diabetes resulted in decreased resting HR in SD
animals compared with SC (Table 1). Resting HR did not differ between ED and EC
rats. The swimming training program was capable of reducing the resting HR for the
EC group when baseline and end-study comparisons were made.
Diabetes was associated with interstitial fibrosis by the accumulation of
myocardial collagen (Fig. 2 and 3). Collagen content was significantly greater in SD
rats compared with SC (7.41 ± 2.08% vs. 3.04±0.26%, respectively; p < 0.05) and in
ED compared with EC rats (4.29 ± 0.36 vs. 2.82±0.38, respectively; p < 0.047) (Fig.
1 and 2). Interestingly, the exercise training reduced collagen accumulation in the LV
of diabetic animals (SD vs. ED, p < 0.05). However, this was not true in the
nondiabetic animals (Fig. 1, 2 and 3).
LV Total collagen (%)
10
Sedentary
Exercised
*
8
6
#†
4
2
0
Contol
Diabetic
Figure 1. Exercise prevented the accumulation of myocardial collagen. Left
ventricular total collagen content expressed as mean ± SD in all four experimental
groups. Statistics: *, significant difference from sedentary control (SC); †, significant
difference from exercised control (EC); #, significant difference from sedentary
diabetic (SD). LV: left ventricle; magnification, ×200.
21
Figure 2. Representative image of the myocardium stained with Sirius Red, observed
under a polarizing microscope. (A) Sedentary control, (B) exercised control, (C)
sedentary diabetic, and (D) exercised diabetic groups. Arrows = collagen fibers.
Arrows pinpoint areas filled with collagen. Observe the increase of collagen fibers in
panel C and its less collagen formation in panel D for those submitted to 8 weeks of
swimming training. Asterisks = interstitial fibrosis; magnification, ×200; bar: 15 µm.
22
Figure 3. Diabetes-induced cardiac fibrosis. The red color of Sirius Red staining
under light microscopy indicates total collagen deposition. (A) Sedentary control, (B)
exercised control, (C) sedentary diabetic, and (D) exercised diabetic groups. Arrows
= collagen fibers. Observe the increased amount of collagen fibers in panel C and its
reduction (near normality) in panel D. Asterisks = interstitial fibrosis; magnification,
×400; bar: 30 µm.
Qualitative analysis of the myocardium using PAS and Gomori’s reticulin
staining is summarized in Table 2. The sarcoplasm and endomysium of the LV of
diabetic sedentary rats had a more prominent staining for polysaccharides than the
exercised diabetic (Fig. 4). Animals from the ED group showed a PAS-positive
reaction partially reduced in sarcoplasms and in endomysium compared with the EC
group. Moreover, the accumulation of PAS-positive material was not observed in
amylase-treated sections (negative controls: Fig. 4E and F).
23
Table 2. Qualitative analysis of the LV of rats by Periodic acid-Schiff (PAS) and
Gomori’s reticulin histochemical techniques
Histochemistry techniques
SC (n = 5)
EC (n = 5)
SD (n = 5)
ED (n = 5)
PAS-sarcoplasm
+
+
++++
+++
PAS-endomysium
+
+
++++
++
Gomori’s reticulin
+
+
+++
++
PAS staining and Gomori’s reticulin staining score: (+) weak reaction with the
staining technique, (++) moderate reaction with the staining technique, (+++) strong
reaction with the staining technique, (++++) intense reaction with the staining
technique. (A) Sedentary control, (B) exercised control, (C) sedentary diabetic, and
(D) exercised diabetic groups.
The silver impregnation of reticular fibers by Gomori’s reticulin technique
demonstrated the fine structure of myocardial collagen, observed as delicate
meshworks of fine fibrils stained black by the silver impregnation. This technique
demonstrated that there were differences among the contents of reticular fibers in
diabetic animals (Fig. 5C and D) compared with nondiabetic (Fig. 5A and B).
Animals from the SD group (Fig. 5C) had greater silver impregnation reaction
compared with animals from the SC group (Fig. 5A). Similarly, the ED animals (Fig.
5D) presented a more intense reaction than those in the EC group. When comparing
the diabetic groups, it appears that exercise training was capable of reducing the
occurrence of reticular fibers in these animals.
24
Figure 4. Periodic acid-Schiff (PAS) staining for polysaccharides. Qualitative
analysis shows glycogen accumulation and PAS-positive staining in cardiomyocytes
after 8 weeks of exercise training. (A) Sedentary control, (B) exercised control, (C)
sedentary diabetic, and (D) exercised diabetic groups. In the groups in panel E
(sedentary diabetic) and panel F (exercised diabetic), note the absence of glycogen in
the sarcoplasm in amylase-treated sections (negative controls). Arrows indicate
intracellular glycogen, and asterisks indicate the PAS-positive endomysium.
Magnification, ×1,000; bar: 20 µm.
25
Figure 5. Silver-impregnated section of myocardium subjected to the Gomori’s
reticulin technique. (A) Sedentary control, (B) exercised control, (C) sedentary
diabetic, and (D) exercised diabetic groups. Arrows = reticular fibers consisting
mainly of collagen type III. Observe the increase of reticular fibers in panel C and its
reduction in panel D. Note in panel C the reticular fibers associated with
cardiomyocyte degeneration and possibly a process of recovery from injury.
Asterisks = thick collagen fibers; magnification, ×1,000; bar: 20 µm.
Discussion
The present study evaluated the effects of low-intensity swimming training on
the structural remodeling of the LV in growing rats with untreated experimental
diabetes. Our data showed that the STZ dosage used induced DCM as indicated by
morphological changes such as increased interstitial collagen, increased fine reticular
fiber, and the accumulation of glycogen in the LV’s extracellular collagen matrix.
Interestingly, part of the adverse cardiac remodeling was attenuated in response to
26
chronic exercise, as observed by reduced collagen deposition and the accumulation
of glycogen.
We found increased LV collagen deposition on STZ-induced diabetic rats,
which is in agreement with previous studies [2,4,13,32,33]. Our results showed that
the degree of total collagen deposition in SD rats was significantly more intense, and
consequently, fibrosis was more evident compared with normal rats. These data
substantiates a histomorphological remodeling process, which is able to endanger the
heart function. In fact, collagen deposition leads to a stiffer myocardium, and
consequently, diastolic dysfunction is observed [12]. Even though this was not
directly evaluated in this study, based on collagen findings, it is possible that diabetic
animals have developed diastolic dysfunction as observed by Loganathan et al. [35].
Additionally, the degree of interstitial fibrosis in ED rats was reduced
compared with the SD group. Cardiac fibrosis is characterized by the proliferation of
cardiac fibroblasts and the excessive accumulation of matrix proteins, mainly
collagen types I and III, in the extracellular space [31,56,60]. This occurs due to
imbalanced ECM metabolism, characterized by increased collagen synthesis and
decreased collagen degradation [56]. Moreover, glycated proteins can undergo a
series of chemical rearrangements to form complex compounds and cross-links
known as advanced glycation end products (AGEs) [40]. The accumulation of AGE
in collagen was associated with reduced collagen turnover, increased cross-linking of
collagen, and stiffness of arteries and myocardium [40]. Furthermore, collagen
glycation increases the formation and migration of myofibroblasts in the heart, a
critical event during fibrosis development in diabetes [60].
Our study demonstrated LV fibrosis in growing diabetic rats, and similar
changes were demonstrated by others either in young and adult rodents [31] or in
humans [60]. Interstitial and perivascular fibrosis has been described in the
myocardium of animals and patients with DM [27]. Myocardial fibrosis is the major
hypothesis for the pathogenesis of DCM [2,6]. Thus, after prolonged hyperglycemia,
the ECM of the cardiac interstitium can be profoundly affected in diabetes [31,60],
which may manifest as increased cross-linking of collagen and alteration of its
functional properties [60]. Currently, the understanding of the effects of type 1 DM
on the morphological characteristics in the myocardium of pediatric populations is
vague, although there are evidences that short-term diabetes in the otherwise-healthy
27
child and adolescent can produce myocardial dysfunction that may be a threat to the
development of severe cardiomyopathy at adulthood [7].
Our qualitative analysis of PAS staining demonstrated a greater tissue
distribution of polysaccharides in the LV myocardium of diabetics compared with
control animals. The accumulation of PAS-positive material was also observed in the
sarcoplasm of cardiomyocyte of the SD rats, which is in concordance with previous
studies [13,36,43].
The effects of diabetes on myocardial glycogen metabolism are directly related
to decreased glucose transport, which might result in increased myocardial glycogen
[43]. However, the excess of glycogen brings about cardiac structural and
physiological impairments, including changes in pH, ionic imbalances, and
stimulations of pathways leading to hypertrophic signaling [45]. It is known that
energy metabolism is rapidly shifted in the diabetic metabolism, resulting in
augmented fatty acid and decreased glucose consumption [21]. The switch of cardiac
energy substrate utilization from carbohydrate to lipids increases intracellular
glycogen, probably through increased glycogen synthesis or impaired glycogenolysis
or a combination of both [24]. Therefore, the greatest intensity in the PAS-positive
reaction observed in the LV of sedentary diabetic rats in the present study may be
related to glycogen stored and spared in the myocardium [13]. In addition, a higher
reaction found in the endomysium by PAS staining may be related to morphological
changes involved in the mechanisms of type III and type IV collagen deposition,
both positive to PAS technique [13,19].
Although exercise seemed to improve accumulation of glycogen in our
trained animals, diabetes played its role. This was demonstrated by a slightly greater
reaction of PAS-positive material in the sarcoplasm of the exercised diabetic rats
when compared with the nondiabetic animals. These results highlight the benefits
that the exercise training exerted on these animals by partially recovering the
polysaccharides tissue distribution. According to Castellar et al. [13], this recovery
might be attributed to an improved metabolic status provided by physical exercise,
which apparently reduces the necessity for glycogen production.
In this study, histochemistry by Gomori’s reticulin staining showed reticular
fibers surrounding the cardiomyocytes, including the endomysium with a more
intense stain reaction in the diabetics groups. Our histological and histochemical
analyses confirm the remodeling of the interstitial matrix demonstrated by increased
28
total collagen deposition in the ECM of diabetic animals reported previously [31].
This adaptation was reduced by exercise training (ED animals) and was unchanged
in nondiabetic animals (SC and EC). However, the reduction of reticular fiber in ED
animals is partially different from previous reports [13]. Castellar and colleagues
[13] found only a slight increased stain reaction to silver impregnation in sedentary
diabetic rats compared with exercised diabetic and control animals. These
controversial results could be attributed to differences in exercise training protocols
or even to age differences among these studies. In this study, the duration of the
training sessions was 30 min longer, and the animals were 40 days younger than
those of Castellar’s protocol [13]. A higher reaction of the endomysium by Gomori’s
reticulin staining in our study also may be involved in the mechanisms of type III
collagen deposition, corroborating with our data of PAS staining [13]. Though the
interactions of ECM components with silver may not be specific, they can
nevertheless provide important insights into the mechanisms of the chemical
reactions involved in ECM remodeling [57]. However, the precise mechanism for the
protection associated with decreased reticular fiber by exercise in our study is
unknown.
Fasting plasma glucose at rest was not affected by the swimming program in
the current study. These results are consistent with other reports [34,35,50]. It is
likely that there has been an improvement in glucose uptake in ED animals. Glucose
uptake into cardiomyocyte occurs through GLUT1 and GLUT4 transporters [1].
Studies have demonstrated that AMP-activated protein kinase (AMPK) promotes
GLUT4 redistribution to the sarcolemmal membrane, improving glucose capitation
[1]. Moreover, it is possible that there has been an increase in glucagon secretion in
ED animals, and its counterregulatory action has helped maintain hyperglycemia
[50]. Thus, a possible improvement of glucose uptake may have been able to prevent
the morphological changes demonstrated in diabetic animals. However, studies have
demonstrated that exercise was able to improve glucose metabolism in diabetic rats
with the reduction of blood glucose levels [5,23]. In agreement with Chimen et al.
[15], there is poor evidence for a beneficial effect of physical activity on glycemic
control. Nevertheless, there are evidences to recommend physical activity in the
management of type 1 DM [15], although the duration and intensity of exercise,
especially for the young, needs further clarification.
29
In the current study, we found that diabetes in rats resulted in decreased HR at
rest. It was surprising that we found no statistical differences in the HR between
exercised and sedentary diabetic animals. Bradycardia has been reported previously
in adult rats [16,52] and adolescent [37] with type 1 DM. Thus, the decreased HR
observed in our study might be associated with autonomic dysfunction. Bradycardia
is a very early indication of DCM [52]. Furthermore, cardiovascular autonomic
neuropathy in diabetes commonly leads to abnormalities in HR control and vascular
dynamics [39]. Additionally, in rats, degenerative changes in autonomic neurons can
be observed from 3 days to several weeks after STZ injection [16].
Conversely, studies have reported that exercise training prevents cardiac
autonomic nervous dysfunction in diabetics [15,59] and reverses bradycardia [16].
Diabetic rats were exercised at a low intensity during 7 weeks, and there was a
significant decrease in resting and post-stress test HR [52]. However, further
investigation is needed to clarify the causes of the lower heart rate in young rats with
type 1 DM.
Conclusion
We concluded that low-intensity swimming training attenuated total collagen
deposition on the ECM and the accumulation of glycogen in the LV of growing rats
with untreated severe experimental diabetes. Our results support the idea that this
type of regular physical activity plays a beneficial role in the adverse remodeling of
the myocardium in rats with type 1 DM. Further studies focusing on the metabolic
disorders associated with diabetes in the youth are required.
Acknowledgements
The authors are grateful to the Fundação de Amparo à Pesquisa de Minas GeraisFAPEMIG (FAPEMIG) for the financial support (process CDS - APQ-01171-11/
PRONEX). AJ Natali is a CNPq fellow.
30
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37
Artigo 2
Swimming training attenuates the morphological reorganization of the
myocardium and local inflammation in the left ventricle of growing rats with
untreated experimental diabetes
Edson da Silvaa,b, Antônio José Natalic*, Márcia Ferreira da Silvaa, Gilton de Jesus
Gomesc,e, Daise Nunes Queiroz da Cunhac, Marileila Marques Toledod, Filipe Rios
Drummondc, Regiane Maria Soares Ramosc, Eliziária Cardoso dos Santosa, Rômulo
Dias Novaesa,f, Leandro Licursi de Oliveiraa, Izabel Regina dos Santos Costa
Maldonadoa*≠
*
a
The authors share senior authorship.
Department of General Biology, Federal University of Viçosa, Viçosa, MG, Brazil.
b
Department of Basic Sciences, Federal University of Jequitinhonha and Mucuri
Valleys, Diamantina, MG, Brazil.
c
Department of Physical Education, Federal University of Viçosa, Viçosa, MG,
Brazil.
d
Department of Medicine and Nursing, Federal University of Viçosa, Viçosa, MG,
Brazil.
e
Department of Physical Education, Federal University of Jequitinhonha and Mucuri
Valleys, Diamantina, MG, Brazil.
f
Department of Biological Sciences and NUPEB, Federal University of Ouro Preto,
MG, Brazil.
Keywords: Adiponectin; diabetes mellitus; ventricular remodeling; exercise.
≠
Address for correspondence: Izabel Regina dos Santos Costa Maldonado,
Departamento de Biologia Geral, Universidade Federal de Viçosa, Edifício Chotaro
Shimoya, Avenida PH Rolfs, s/nº, CEP: 36.570-000 Viçosa, Minas Gerais, MG,
Brazil. Tel: +55 (031) 3899-3365. E-mail: [email protected]
38
Abstract
Diabetic cardiomyopathy is associated not only with cardiac remodeling and
myocardial dysfunction but also with the occurrence of low-grade inflammation and
reduced cardiac adiponectin resulting in significant morbidity and mortality in
patients with type 1 diabetes mellitus (T1DM). On the other hand, physical exercise
is an important strategy for the management of diabetes which can reduce
inflammation and attenuate adverse cardiac remodeling and contractile dysfunction.
The aim of this study was to investigate the influence of low-intensity swimming
training
in
cardiac
cytokines,
inflammation,
structural
remodeling,
and
cardiomyocyte contractile dysfunction in growing rats with untreated experimental
T1DM. Thirty-day-old male Wistar rats were divided into four groups (n = 14, per
group): sedentary control (SC), exercised control (EC), sedentary diabetic (SD), and
exercised diabetic (ED). Diabetes was induced by streptozotocin (STZ, 60 mg kg-1
body weight). Animals from EC and ED groups were submitted to a swimming
training (5 days/week, 90 min/day, load of 5% body weight) for 8 weeks. After
euthanasia, the left ventricle (LV) was removed for molecular, morphological, and
cardiomyocyte mechanical analysis. Diabetic animals presented cardiac remodeling
with myocardial histoarchitectural disorganization, fibrosis, inflammatory infiltrate
and necrosis. The capillary density was significantly lower in diabetic animals. Left
ventricular cardiomyocytes from diabetic animals exhibited more prolonged time to
the peak of contraction and the time to half relaxation than those from control
animals, but no difference in cell shortening was observed. The cardiac levels of
interleukin 10, nitric oxide, total and HMW adiponectin were significantly decreased
in diabetic animals. Exercise training reduced the level of TNF-α, increased capillary
density and attenuated the histopathological parameters assessed in the LV of
diabetic rats. In conclusion, the adverse cardiac structural remodeling induced by
experimental T1DM coexists with reduced levels of total and HMW adiponectin,
chronic inflammation and cardiomyocyte contractility dysfunction. More important,
low-intensity swimming training attenuated part of these pathological changes which
indicate the beneficial role for regular exercise untreated T1DM.
39
Introduction
Diabetes mellitus (DM) is a group of metabolic diseases characterized by
hyperglycemia resulting from defects in insulin secretion, insulin action, or both
(American Diabetes Association, 2013) with high prevalence in both adults and
adolescent populations (Ma and Chan, 2009). Cardiovascular disease (CVD) is one
of the major complications of diabetes that commence in childhood (Nadeau et al.,
2011) and greatly impacts mortality and morbidity (Guo et al., 2007; Rajesh et al.,
2012).
Type 1 DM (T1DM) is primarily a disease of insulin deficiency from
pancreatic beta-cell destruction (American Diabetes Association, 2013) and CVD
may be linked to insulin resistance in this form of diabetes (DeFronzo et al., 1982;
Pereira et al., 2012). Moreover, DM has been related to low-grade inflammation and
micro- and macrovascular complications in adults (Gonzalez-Clemente et al., 2007;
Llauradó et al., 2012) and children with T1DM (Mangge et al., 2004).
Low-grade inflammation has been associated with an increase proinflammatory circulating proteins, such tumor necrosis factor-alpha (TNF-α),
interleukin (IL-6), C-reactive protein (CPR) and with a reduction in the antiinflammatory proteins such as IL-10 (Ryba et al., 2011; Llauradó et al., 2012). A
chronic inflammation status leads to histopathological changes with tissue injury,
endothelial dysfunction, and cardiac remodeling resulting in deterioration of
myocardial contractility and left ventricular function (Llauradó et al., 2012). The
suggested mechanisms are through the reduction of Ca2+ transient (Duncan et al.,
2007) and reduction of Ca2+ sensitivity of the contractile myofilaments (Goldhaber et
al., 1996).
Adiponectin also majorly impacts the pathogenesis of insulin resistance,
diabetes and vascular injury (Gu and Li, 2012; Fenger, 2013), and plays an important
role in glucose and lipid metabolism (Numao et al., 2008; Garekani et al., 2011;
Pereira et al., 2012). Adiponectin is a plasma protein mainly secreted by adipocytes
(Bobbert et al., 2011) with anti-diabetic, anti-inflammatory, antiatherogenic
properties (Garekani et al., 2011; Forsblom et al., 2011). Moreover, cardiomyocytes
are also capable of synthesizing adiponectin (Piñeiro et al., 2005; Maia-Fernandes et
al., 2008). Studies suggest that, adiponectin, in particular, the high-molecular weight
(HMW) adiponectin isoform (Leth et al., 2008; Numao et al., 2008), is a potent
immunomodulatory and cardioprotective molecule (Essick et al., 2011; Jenke et al.,
40
2013). In fact, this protein protects the heart from ischemic injury, cardiomyopathy
and
systolic
dysfunction
(Goldstein et
al.,
2009),
inhibits
pathological
cardiomyocyte hypertrophy and myocardial fibrosis (Han et al., 2007; Maia-Ferandes
et al., 2008), reduces oxidative and nitrative stress (Goldstein et al., 2009; Wang et
al., 2013), reduces TNF-α and IL-6, and increases expression of IL-10 in the heart
(Han et al., 2007; Maia-Ferandes et al., 2008; Goldstein et al., 2009).
Adiponectin improves cardiomyocyte contractile function in db/db diabetic
obese possibly by alleviating endoplasmic reticulum stress (Dong and Ren, 2009). In
addition, in diabetes, endothelial nitric oxide synthase (eNOS) protein expression is
progressively reduced in myocardium and nitric oxide (NO) content is decreased
(Wang et al., 2011; Wang et al., 2013, Nagareddy et al., 2005).
Regular physical exercise is an important strategy for the management of
T1DM, due the beneficial health effects, especially cardiovascular disease prevention
(Galassetti and Riddell, 2013). Aerobic exercise training can reduce inflammation
and cardiovascular risks (Rosa et al., 2010; Galassetti and Riddell, 2013), reduces
insulin resistance, improves glucose and lipid metabolism (Khan, 2013), attenuates
morphological changes (Silva et al., 2013) and contractile dysfunction in both
animals and human with DM (Bidasee et al., 2008; Chimen et al., 2012; Rajesh et al.,
2012).
The regulation of glucose or lipid metabolism by adiponectin through
exercise training have been investigated (Ando et al., 2009; Garenaki et al., 2011),
however, inconsistent findings have emerged, mainly on total circulating and HMW
adiponectin levels (Ando et al., 2009).
So far, few studies have examined the relationship of factors influencing
adiponectin levels in children and adolescents with type 1 DM, and the results of
those studies are very inconsistent (Huerta, 2006; Habeeb et al., 2012; Menon, 2012;
Karamifar et al., 2013). In addition, recent studies showed reduction in the cardiac
adiponectin expression in STZ-induced diabetic rats (Pei et al., 2013). However, the
relationship between cardiac adiponectin in growing rats with T1DM and the effects
of a low-intensity swimming training has not been investigated yet.
Therefore, we hypothesized that low-intensity swimming training can reduce
the effects of STZ-induced diabetes on the cardiac histopathology, cytokines, NO,
capillary density as well as on the cardiomyocyte contractile dysfunction.
41
Materials and Methods
Animals and experimental groups
Male Wistar rats weighing 90.0 ± 5.0 g, 30 days old, were obtained from the
animal facility at the Federal University of Viçosa, Brazil, and were randomly
divided into four groups (n = 14, per group): sedentary control (SC), exercised
control (EC), sedentary diabetic (SD), and exercised diabetic (ED). Rats were
maintained on 12-h dark/light cycle at 22°C, humidity at 60–70%, housed in groups
of five, and fed standard commercial rodent chow and water ad libitum. All
procedures followed the Guidelines for Ethical Care of Experimental Animals and
were approved by the Ethics Committee on Animal Experimentation (CEUA) from
the Federal University of Viçosa (protocol number 46/2011).
Diabetes induction
Severe diabetes was induced in the animals by intraperitoneal injection of
streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO) dissolved in 0.1 M citrate buffer
solution (0.1 M, pH 4.5) at the dose of 60 mg kg-1 body weight (BW) (Howarth et al.,
2010). Equivalent volume (1 mL kg-1) of vehicle was injected into the rats assigned
to the control groups. Animals were fasted overnight for 12 h prior to STZ
administration. Water and food were available immediately after dosing. Diabetes
was determined seven days after STZ injection. Glycemia (> 300 mg/dL) (Siu et al.,
2006) was dosed using the Accu-Chek Advantage glucometer (Boehringer
Mannheim Corporation, Indianapolis, IN) after a fasting period of 12-hours
overnight. Body weights and blood glucose levels were recorded once a week
throughout the study. All animals were euthanatized 8 weeks after diabetes induction
by intraperitoneal injection of sodium pentobarbital (120 mg kg-1).
Exercise protocol
After seven days of diabetes induction and confirmation of consistent
hyperglycemia, animals from the exercised groups (ED and EC) were submitted to a
swimming training program (adapted from Gomes et al., 2006) for 8 weeks. Rats
were placed in water tank measuring 65 cm in height and 75 cm in diameter, filled
with warm water (28°C to 30°C) at a depth of 45 cm and forced to swim. Animals
were then dried and returned to the home cage. Training intensity varied by changing
42
a load that was placed around the animal’s chest from 0% to 5% of its BW. Briefly,
in the first week, animals exercised in the water for 10 to 50 min, with no load, while
duration was increased by 10 min each/day. In the second week, animals remained
exercising with no load and with the duration incremented by 10 min/day until a
maximum of 90 min of continuous swimming. From the fourth week, animals began
swimming with a load until the end of the training program (8 weeks). The load was
progressively increased by 1% of the animal’s BW from the fourth week on, such
that at the eighth week, animals swam with a total load of 5% of their BW. During
the swimming sessions, animals from the sedentary group (SD and SC) were placed
in a polypropylene box containing warm water (28°C to 30°C) with a depth of 10
cm.
Electrocardiogram
Four animals per group randomly selected were anesthetized in an induction
chamber flushed with 2% isoflurane and 100% oxygen at a constant flow of 1 L min1
. Once unconscious, they were placed on a platform in dorsal recumbency, with the
four limbs fixed. Isoflurane was maintained at a concentration sufficient for restrain
(0.5% to 1.0%), and animals were able to maintain spontaneous breathing during the
electrocardiogram (ECG). A trichotomy of approximately 1 cm2 was performed in
the forelimbs and left hindlimb for electrode insertion. Derivation II of the ECG was
recorded using the data acquisition system PowerLab ® (AD Instruments, São Paulo,
Brazil), and data analysis was done with the program LabChart Pro ® (ADInstruments
LabChart 7, São Paulo, Brazil).
Electrocardiograms were performed at the end of the experiments by an
experienced fellow blind to the study groups and treatments. Resting heart rate (HR)
was derived from the ECG, following established guidelines (Heffernan and Jae,
2007).
Histological processing and histochemistry
Following euthanasia, the heart of five animals per group randomly selected
was excised, washed with saline solution, dissected and weighed separately. The LV
was dissected and weighed separately. Half of the LV tissue was rapidly frozen in
liquid nitrogen and stored at -80° C for subsequent assay, and the other half of the
LV was fixed
by imm ersion in 4% paraformaldehyde in 0.1 M sodium
phosphate buffer,
43
pH 7.2–7.4, for 24 h. Left ventricle fragments were obtained through the orientator
method to define isotropic and uniform random sections (IUR) required in the
stereological study (Mandarim-de-Lacerda et al., 2003). These fragments were
dehydrated in ethanol, cleared in xylol, and embedded in paraffin or
glycolmethacrylate resin (Historesin® – Leica). Blocks were cut into 5 μm or 2 μm
sections and mounted on histological slides. The LV sections were stained by Sirius
red (for total collagen), Gomori's trichrome (for microcirculation), and toluidine
blue/sodium borate 1% to evaluate the series of histopathological changes in the
diabetic myocardium. Digital images were captured using a light microscope (mod.
Primo Star, Carl Zeiss AG, Oberkochen, Germany) connected to a digital camera
(AxioCam ERc5s, Carl Zeiss AG).
Analysis of myocardial microcirculation
To quantify intramyocardial capillaries, LV was evaluated in histological
sections 5 µm thick stained with Gomori's trichrome. The distribution of capillary
density was analyzed using the image analysis software Image Pro-Plus 4.5® (Media
Cybernetics, Silver Spring, MD, USA). In this analysis, 12 microscopic fields from
five rats per group were randomly investigated (magnification ×400). A 208-point
grid was superimposed on each image, and the total of 2,496 points were assigned
for each rat. The area was determined by adding up these points, then dividing the
result by the total points for the cross-section. Results were expressed as mean ±
SEM.
Sirius red and toluidine blue/sodium borate staining
Pathological analysis of the left ventricle was performed using toluidine
blue/sodium borate 1% and Sirius red stain. In this analysis, 12 microscopic fields
from five rats per group were randomly investigated (magnification ×400). Tissue
sections of LV were stained with Sirius red for detection of collagen accumulation,
perivascular fibrosis and interstitial fibrosis, and toluidine blue for general
pathological alterations.
ELISA-based cytokine detection assay
The levels of Interleukin 10 (IL-10), Tumor Necrosis Factor alpha (TNF-α),
adiponectin and High molecular weight (HMW) adiponectin were measured from the
44
LV homogenate by ELISA. Cytokines IL-10, TNF-α and adiponectin were assayed
using Uscn life science Inc. (Wuhan in China) kits and high molecular weight
(HMW) adiponectin from BioVendor Co. (Heidelberg, German). The ELISA
procedure was performed according to the manufacturer’s protocol. The cytokine
concentrations were determined with reference to a standard curve for serial two-fold
dilutions of the rat recombinant cytokines.
NO Production
NO production was quantified by the standard Griess reaction (Sambrook and
Russell, 2001). Briefly, 50 µl of supernatants from the left ventricle homogenates
were incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1%
naphthalene diamine dihydrochloride, and 2.5% phosphoric acid) at room
temperature, for 10 minutes. The absorbance was measured at 550 nm in a
microplate scanning spectrophotometer (Power Wave X, Bio-Tek Instruments, Inc.,
Winooski, VT). The conversion of absorbance into micromolar concentrations of NO
was deduced from a standard curve using a known concentration of sodium nitrite.
Cardiomyocyte isolation
Nine animals from each group were used to cardiomyocyte contractile
function measurement. Two days after the last training session, the rats were
weighed and euthanized by cervical dislocation under resting conditions, and their
hearts were quickly removed. Left ventricular myocytes were enzymatically isolated
as previously described (Natali et al., 2002). Briefly, the hearts were mounted on a
Langendorff system and perfused for ~5 min with a modified Hepes–Tyrode solution
of the following composition (in mM): 130 NaCl, 1.43 MgCl2, 5.4 KCl, 0.75 CaCl2,
5.0 Hepes, 10.0 glucose, 20.0 taurine, and 10.0 creatine, pH 7.3 at 37 °C. The
perfusion solution was changed for the calcium-free solution with EGTA (0.1 mM)
for 6 min. Afterwards, the hearts were perfused for 15–20 min with a solution
containing 1 mg mL-1 collagenase type II (Worthington, USA). The digested heart
was then removed from the cannula, and the ventricles were removed and weighed.
The left ventricle was separated, weighed, and cut into small pieces. The left
ventricle fragments were placed into small conical flasks with collagenase-containing
solution supplemented with 1% bovine serum albumin. The cells were dispersed by
agitating the flasks at 37 °C for periods of 5 min. Then, single cells were separated
45
from the non-dispersed tissue by filtration. The resulting cell suspension was
centrifuged and resuspended in Hepes–Tyrode solution. Non-dispersed tissue was
subjected to further enzyme treatment. The isolated cells were stored at 5 °C until
use. Only calcium-tolerant, quiescent, rod-shaped cardiomyocytes showing clear
cross-striations were studied. The isolated cardiomyocytes were used within 2–3 h of
isolation.
Measurements of cell contractility
Cell contractility was evaluated as previously described (Roman-Campos et
al., 2009). Briefly, isolated cells were placed in a chamber with a glass coverslip base
mounted on the stage of an inverted microscope (Nikon Eclipse-TS100, USA). The
chamber was perfused with Hepes–Tyrode solution at 37 °C. Steady-state 1 Hz
contractions were elicited via platinum bath electrodes (Myopacer, Field Stimulator,
Ionoptix, USA) with 5 ms duration voltage pulses and an intensity of 20 V. Cells
were visualized on a PC monitor with a NTSC camera (Myocam, Ionoptix, USA) in
partial scanning mode. This image was used to measure cell shortening (our index of
contractility) in response to electrical stimulation using a video motion edge detector
(IonWizard, Ionoptix, USA). The cell image was sampled at 240 Hz. Cell shortening
was calculated from the output of the edge detector using an IonWizard A/D
converter (Ionoptix, Milton, MA, USA). Cell shortening (expressed as a percentage
of resting cell length), time to peak shortening and time to half relaxation were
calculated.
Statistical analysis
Results were expressed as mean ± SEM. The Kolmogorov-Smirnov test for
normality was initially performed. Statistical significance differences were evaluated
by ANOVA one way on Ranks followed by the Dunn's test. Two-way ANOVA or
unpaired t-test was used when appropriate, and the post hoc Tukey test was applied
for multiple comparisons. The analysis was performed using the Sigma Stat software,
version 3.0, and statistical significance was defined as p ≤ 0.05.
Results
The animals from SD and ED groups presented the expected hyperglycemia
found in severe diabetes. At the baseline, there were no differences in blood glucose
46
(BG) levels between diabetic (SD vs. ED) and control rats (SC vs. EC) (Table 1).
Blood glucose increased throughout the experiment in both SD and ED rats. Seven
days after the application of STZ and at the end of the study, the glycemic levels for
the diabetic groups (SD and ED) were significantly greater (Table 1). Swimming
training reduced the BG level by 34 mg dL-1 in the diabetic groups, however this was
not statistically different (p = 0.16).
Mean body weight at baseline was similar across all groups (Table 1).
Diabetic rats had lower BW gain compared with normal rats (SC and EC). Similarly,
the LV weighted significantly less in the diabetic rats (SD and ED). Streptozotocin
induced diabetes increased the index of LV hypertrophy (VW/BW ratio) observed in
SD vs. SC and ED vs. EC rats (Table 1). The index of hypertrophy did not differ
between ED and SD rats (Table 1).
Table 1. Biometrical and functional parameters
Parameters
SC
EC
SD
ED
90.80 ± 3.37
89.40 ± 5.01
86.20 ± 5.67
91.20 ± 6.87
Final BW, g
320.60 ± 26.05
334.60 ± 13.57
164.60 ± 5.89*
170.60 ± 5.18†
WG, g
229.80 ± 25.42
245.20 ± 13.17
75.20 ± 7.69*
79.40 ± 4.76†
VW, mg
339.20 ± 17.45
379.20 ± 19.09
211.80 ± 6.67*
222.40 ± 4.04†
VW/BW, mg g1
1.06 ± 0.30
1.13 ± 0.63
1.29 ± 0.51*
1.30 ± 0.35†
Baseline BG, mg dL1
73.20 ± 5.35
68.20 ± 5.47
74.20 ± 4.33
73.60 ± 8.54
BG Post-STZ, mg dL1
72.60 ± 3.31
65.40 ± 2.48
429.20 ± 33.65*
461.18 ± 11.07†
Final BG, mg dL1
98.20 ± 7.93
89.80 ± 2.71
567.80± 19.78*
533.00 ± 6.25†
Resting HR, bmp
356.40 ± 7.39
300.50 ± 20.44*
262.98 ± 7.39*
274.35± 20.53
Baseline BW, g
Data are presented as mean ± SEM (Resting HR, four animals per group, other
parameters n = 14 animals per group); SC, sedentary controls; EC, exercised
controls; SD, sedentary diabetics; ED, exercised diabetics; BG, blood glucose; BW,
body weight; VW, ventricular weight; WG, weight gain; HR, heart rate. *, different
from SC; †, different from EC; (p < 0.05).
47
Diabetes caused a decreased resting HR in SD vs. SC rats (p < 0.05).
However, resting HR did not differ between animals that exercised (Table 1). The
swimming training program reduced the resting HR for the EC group when
compared to SC measurements.
As shown in Fig. 1, total adiponectin tissue levels were significantly lower in
the diabetic rats (SD 3.13 ± 0.13 and ED 3.41 ± 0.38) compared with controls (SC
4.92 ± 0.25 and EC 5.64 ± 0.26). Similarly, the HMW adiponectin level was
significantly lower in the SD vs. SC (1.78 ± 0.01 and 3.01 ± 0.02, respectively) and
ED vs. EC (2.19 ± 0.01 and 3.41 ± 0.02, respectively) rats. Swimming training
program promoted a non-significant increasing trend in the levels of HMW
adiponectin in the EC (p = 0.06) and ED (p = 0.07) animals.
Tissue levels of IL-10 were significantly lower in the diabetic rats (SD 0.21 ±
0.01 and ED 0.41 ± 0.15) compared with controls (SC 0.78 ± 0.06 and EC 1.43 ±
0.32). Diabetes increased TNF-α levels in the LV of diabetic rats (SD and ED)
compared with control rats (SC and EC, respectively). Exercise promoted a nonsignificant increasing trend in the IL-10 levels in both group, control (p = 0.08) or
diabetic animals (p = 0.09) (Figure 1C), and reduced TNF-α levels in the diabetic
group (Fig. 1d).
48
Figure 1. ELISA assay expression of cytokines in the LV of control and diabetic
rats. Graphs show the quantitative levels of (a) total adiponectin, (b) HMW
adiponectin, (c) IL-10, and (d) TNF-α of the indicated sample groups. Quantitative
data are displayed as mean ± SEM (n = 5 animals per group). *Significant difference
from sedentary control (SC). †Significant difference from exercised control (EC).
#
Significant difference from sedentary diabetic (SD) (p < 0.05).
Capillary density analysis of the LV showed that diabetic animals presented a
lower total capillary density compared SC animals (Fig. 2a-d and 2e, p < 0.05).
Capillary density of the LV was greater in the ED and EC groups compared to SD
and SC, respectively, and ED vs. SD (Fig. 2e). Diabetes was associated with reduced
levels of myocardial nitric oxide (Fig. 2f, p < 0.05) compared with the control groups
SC and EC. Interestingly, the exercise training increased NO levels in the LV of EC
rats compared with sedentary control (SC vs. EC; 210.03 ± 19.87 vs. 291.34 ± 13.41,
respectively). Swimming training increased NO level by 31% in the diabetic group
(SD vs. ED), but this did not reach statistical difference (p = 0.11). In addition, a
49
qualitative analysis of the LV revealed greater amounts of collagen fibers around
blood vessels characterizing perivascular fibrosis in SD animals, however this was
found to a lesser degree in ED animals (Fig. 3).
Figure 2. Blood vessels and NO in the LV of control and diabetic rats. Histological
sections of myocardium subjected to the Gomori’s trichrome technique. (a)
Sedentary control, (b) exercised control, (c) sedentary diabetic, and (d) exercised
diabetic groups. Arrows = capillaries. Note the increased level of capillary density in
panel B and its reduction in panels c and d. The arrowhead in panels c and d
indicates cardiomyocyte with vacuolar sarcoplasm (magnification ×400, bar = 30
50
µm). Graphs show capillary density in panel e and the quantitative levels of LV NO
in panel f. Data are displayed as mean ± SEM (n = 5 animals per group). *Significant
difference from sedentary control. †Significant difference from exercised control.
#
Significant difference from sedentary diabetic (p < 0.05).
Figure 3. Diabetes-induced cardiac fibrosis. The red color of Sirius red staining
under light microscopy indicates total collagen deposition. (a) Sedentary control, (b)
exercised control, (c) sedentary diabetic, and (d) exercised diabetic groups. Arrows =
collagen fibers. Note the increased amount of collagen fibers in panel c and its
reduction in panel d. *Perivascular fibrosis (magnification ×400; bar: 30 µm).
In SC and EC rats, cardiomyocytes were well organized, with myofibrils
symmetrically disposed (Figs. 4 a and b). Pathological characteristics from LV of
diabetes were observed. Diabetic LV myocardium demonstrated collagen
accumulation, perivascular fibrosis and intramyocardial fibrosis (Fig. 3 c),
architectural disorganization along with degeneration and atrophy of cardiomyocytes
(Fig. 4 c), and evidence of inflammatory-cell infiltrate (not showed in the figure). In
51
addition, cardiomyocytes from DM rats presented irregular hypertrophy, vacuolar
sarcoplasm, reduction of myofibrils, contraction band necrosis, and nuclei with
irregular shape (Figures 4 c). Pathological characteristics from LV of diabetes were
attenuated by swimming training program (Figs. 3 d and 4 d).
Figure 4.
Histological sections stained with toluidine blue/sodium borate 1%
showing pathological characteristics from the LV of diabetes. (a) Sedentary control,
(b) exercised control, (c) sedentary diabetic, and (d) exercised diabetic groups
(magnification ×400, bar = 30 µm). Asterisks = cardiomyocytes, arrows =
cardiomyocytes nucleus, arrowhead = vacuoles, and star = contraction band necrosis.
Note that pathological characteristics from the LV of diabetes were attenuated by
swimming training in panel d (magnification ×400, bar = 30 µm).
Cardiomyocytes contractility
The analysis of cell contractility showed marked changes in the mechanical
properties of isolated cardiomyocytes from diabetic animals (Figure 5). In LV
cardiomyocytes of animals in the SD group had a significant prolongation of the time
to peak of contraction compared to the SC group (300.93 ± 15.26 ms vs. 231.25 ±
52
7.30 ms, respectively, p<0.05). The swimming training did not reduce the time to
peak contraction in diabetic animals (SD, 300.93 ± 15.26 ms vs. ED, 290.65 ± 4.8
ms, p >0.05). The effect of swimming program was also not detected regarding the
time to peak contraction in LV cardiomyocytes of control animals (EC 227.35 ±
10.93 ms vs. SC, 231.25 ± 7.30 ms, p > 0.05). In addition, SD vs. SC groups (3.05 ±
0.24 and 2.23 ± 0.20, respectively), and ED vs. EC groups (2.52 ± 0.22 and 2.60 ±
0.21, respectively) did not exhibit statistically significant differences in cell
shortening. Time to half relaxation was greater in the SD compared to SC group (SD,
174.26 ± 12.65 ms vs. SC, 146.46±10.06 ms, p < 0.05). The swimming training
program did not affect the time to half relaxation of cardiomyocytes in diabetic
animals (SD, 174.26 ± 12.65 ms vs. ED, 209.78 ± 13.37 ms, p < 0.05). And the same
occurred in the control animals (SC, 146.46±10.06 ms vs. EC, 139.12± 12.22 ms, p <
0.05).
53
Figure 5. Graphs showing the contractility parameters of the LV isolated
cardiomyocytes from control and diabetic rats. (a) Cell shortening expressed as a
percentage of resting cell length, (b) time to peak, and (c) time to half relaxation.
Data are represented as mean ± SEM of 46–68 cells in each group; % r.c.l.,
percentage of resting cell length. *Significant difference from sedentary control.
†
Significant difference from exercised control (p < 0.05); Kruskal-Wallis test
followed by Dunn test.
54
Discussion
In the present study we evaluated the effects of low-intensity swimming
training on the cardiac structural remodeling, cardiac cytokines and cardiomyocyte
contractile function in growing rats with untreated experimental T1DM. The results
showed that STZ-induced diabetes promoted marked myocardial morphological
reorganization and cardiomyocyte contractile function impairment. The left ventricle
of diabetic rats demonstrated pathological characteristics such as histoarchitectural
disorganization,
collagen
accumulation,
fibrosis,
reduced
blood
vessels,
cardiomyocyte with inflammatory infiltrate, necrosis and irregular hypertrophy.
Total adiponectin, HMW adiponectin, IL-10, and NO tissue levels were significantly
lower in diabetes, but LV TNF-α levels were increased. However, our exercise
training protocol induced a trend towards enhanced IL-10 levels, attenuated the TNFα level and the pathological characteristics of the LV in diabetes.
Animals with T1DM presented a marked reduction of total adiponectin and
HMW adiponectin levels in the LV. As far as we know, this is the first study
investigating the levels of cardiac adiponectin in growing rats with diabetes. As of
the three major isoforms of adiponectin, low molecular weight (LMW), middle
molecular weight (MMW), and high molecular weight (HMW) adiponectin
(Hickman and Whitehead, 2012), the HMW is thought to be the major active isoform
in peripheral tissues (Maia-Fernandez et al., 2008, Goto et al., 2013). Consistent with
previous studies on adult animals, the total and HMW adiponectin levels were
inversely associated with T1DM (Pei et al., 2013; Wang et al., 2013). Study by Pei et
al., (2013) demonstrated that cardiac adiponectin levels gradually decreased
throughout T1DM development in adult mice, which was associated with decreased
adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in
diabetic hearts. Wang et al. (2011) showed decreased cardiac adiponectin levels and
increased inflammatory cytokines TNF-α and IL-6 in diabetic rats. This runs parallel
with our results that showed that T1DM rats exhibited reduced cardiac adiponectin
levels and local inflammatory response as compared with the control. In contrast
with our findings, studies performed by Guo et al. (2007) demonstrated that cardiac
adiponectin levels were unchanged in diabetic adult rats, but
the adiponectin
receptor 1 (AdipoR1) was up-regulated in the heart of STZ-induced diabetes (Guo et
al., 2007). Furthermore, increased AdipoR1 expression in cardiomyocyte may be a
55
compensatory mechanism responsible for the cardioprotective effect of adiponectin
in T1DM (Ma et al., 2011; Wang et al., 2013). Despite the increase in AdipoR1
expression, studies showed increased cardiac inflammatory response and decreased
GLUT4 protein expression associated with a reduction cardiac adiponectin
expression (Sakr et al., 2013).
Decreased expression of GLUT4 indicates that
glucose metabolism was reduced in diabetic rat hearts (Guo et al., 2007). In our
study, the inflammatory status and a possible dysfunction of energy metabolism may
be contributed to the deterioration in morphology and cardiac function in STZdiabetic rats which is in agreement with others (Guo et al., 2007; Wang et al., 2011).
In response to swimming training, total and HMW adiponectin were similarly
unchanged in both, diabetic and control groups. These suggest that even if lowintensity aerobic exercise affects the metabolism, probably the production and
secretion of total and HMW adiponectin are not locally affected in the heart of
growing rats. Thus, the effect of exercise training on adiponectin remains to be
elucidated.
In the present study we observed that T1DM caused microvascular damage
and reduced NO levels in the left ventricle. Interestingly, swimming training
increased capillary density. The attenuation of microcirculation impairment in the
ED rats was evidenced by increased capillary density and reduced perivascular
fibrosis. These results are exciting as adiponectin is known to exert protective effects
through its vasodilator, anti-apoptotic, anti-inflammatory, anti-atherogenic and antioxidative activities in both cardiac and vascular cells (Hui et al., 2011; Hickman and
Whitehead, 2012). Our findings demonstrate a reduced density of myocardial
capillaries in T1DM rats which is in concert with previous reports (Lee et al., 2011;
Cosyns et al., 2008). Additionally, a recent study using diabetic rats showed that the
microvascular arrangement was very disordered and the surfaces of cardiac
microvascular walls were highly irregular, with numerous evaginations and
invaginations (Yin et al., 2012). In the current study T1DM affected cardiac levels of
NO as shown previously (Wang et al., 2011; Nagareddy et al., 2005). Evidences
suggest that in T1DM, the impairment of endothelial function may involve reactive
oxygen species such as superoxide that readily react with NO to form peroxynitrate,
which results in decreased NO bioavailability (Heidarianpour, 2010). Wang and
colleagues (2011) demonstrated that STZ-induced diabetes rats displayed reduced
cardiac endothelial nitric oxide synthase (eNOS) activation, which decrease cardiac
56
NO content (Fuchsjäger-Mayrl et al., 2002). This result was associated with reduced
cardiac adiponectin and increased inflammatory cytokines.
We speculated that the reduced levels of both cardiac adiponectin and NO in
the current study could be associated with the mechanisms of left ventricular
microcirculation impairment in T1DM rats because hyperglycemia and oxidative
stress in diabetes can initiate a cardiac structural remodelling
that leads to
deterioration of arteries, capillaries and venules. On the other hand, adiponectin
exerts the protective effects by directly acting on endothelial cells, smooth muscle
cells and macrophages (Ohashi et al., 2012) and also stimulates AMPK activation in
endothelial cells, leading to activation of eNOS (Maia-Fernandes et al., 2008; Ohashi
et al., 2012).
In the present study, the exercise program attenuated significantly reduction
in capillary density and perivascular fibrosis in diabetic rats. These are exercise
benefits to the myocardium inasmuch as the capillary network participates in
maintaining the supply of oxygen and energy substances to the heart. However, in
our study, these changes were independent of adiponectin, suggesting that exercise
plays an effective role in restoring the capillary net in the myocardium of rats with
T1DM via both, the adiponectin-dependent and independent pathways. In addition,
exercise training was capable of stimulating angiogenesis in EC animals compared to
SC animals. These findings are in agreement with those reported by Ellison et al.
(2012) who demonstrated that exercise training induces vascular remodeling of the
cardiac muscle by increasing of capillary density. Exercise training can also decrease
oxidative stress and improve anti-oxidative capacity of the vascular wall, thus it
appears to be beneficial in prevention and improvement of microvascular
dysfunction in T1DM (Heidarianpour et al., 2010). Abnormalities in the
endothelium/NO pathway have been reported in human and animal models of T1DM
in both micro and macro vessels (Khan, 2000; Fuchsjäger-Mayrl et al., 2002).
However, the mechanisms underlying the morphological and functional changes of
blood vessels are complex and only partially understood.
We observed here increased TNF-α and decreased IL-10 levels in the left
ventricle of diabetic rats. In addition, cardiac collagen accumulation (total collagen),
local inflammation, degeneration and atrophy of cardiomyocytes, necrosis and
myocardial fibrosis were also increased in these animals. Interestingly, our exercise
training program attenuated the TNF-α production and left ventricular pathological
57
remodeling in diabetic animals. Our findings are consistent with previous reports in
which intramyocardial inflammation was evidenced by increased levels of cardiac
TNF-α (Westermann et al., 2007; Guo et al., 2007; Rajesh et al., 2012; Wen et al.,
2013; Huang et al., 2013) and decreased levels of cardiac IL-10 (Huang et al., 2013;
Ares-Carrasco et al., 2009). TNF-α is one of the major mediators of inflammation
(Ryba et al., 2011) and endogenous TNF-α plays a central role in initiating and
sustaining the inflammatory response (Kaur et al., 2006; Hui et al., 2011). Cardiac
over-expression of TNF-α has been related to multiple detrimental effects on the
heart, including cardiomyocyte hypertrophy, myocardial contractile dysfunction,
fibrosis, apoptosis, pathologic heart remodeling and reduction of adiponectin, which
leads to heart failure in T1DM (Hui et al., 2011; Rajesh et al., 2012; Nunes et al.,
2012; Wen et al., 2013). A recent study revealed intramyocardial inflammation in
unmanaged diabetic rats seven weeks after STZ injection, as evidenced by enhanced
activity and expression of nuclear factor kappa B (NF-κB), thus leading to increased
levels of cardiac pro-inflammatory cytokines (TNF-α, IL-1β), enhanced expressions
of cell adhesion molecules (ICAM-1, VCAM-1), and activated invading
immunocompetent cells, such as macrophages (CD68+ cells) and T lymphocytes
(CD3+ cells), and cardiac collagen accumulation (Wen et al., 2013). The
consequence of these abnormal structural alterations in diabetic rats lead to impaired
cardiac performance, i.e. increased left ventricular dimensions, reduced ejection
fraction and fractional shortening (Wen et al., 2013). The highest VW/BW of
hypertrophy of diabetic rats in this study reflects the increased dimensions of the LV.
The increase in pro-inflammatory cytokines in diabetes could also have
influenced the levels of cardiac adiponectin and contractile dysfunction in the present
study. Inflammatory cytokines can attenuate cardiomyocyte contractility directly
through the immediate reduction of the Ca2+ transient, via alterations in sarcoplasmic
reticulum function and indirectly through attenuation of myofilament calcium
sensitivity, through nitric oxide-dependent attenuation (Nian et al., 2004; Duncan et
al., 2007; Wen et al., 2013). Sustained expression of TNF-α can also lead to
decreased sarcoplasmic reticulum Ca2+ ATPases (SERCA2) expression, which is
essential for the re-uptake of calcium in an energy-dependent manner after
excitation-contraction of the cardiomyocyte (Nian et al., 2004). However, the
relationship of these molecular changes with myocardial structural remodeling and
58
the cellular inflammatory mechanisms in diabetic cardiomyopathy requires further
investigation.
We also found that TNF-α production was attenuated in the exercised diabetic
animals demonstrating a potential cardioprotective effect provided by exercise
training against future cardiovascular injuries. Evidences support the idea that regular
exercise may suppress proinflammatory cytokines TNF-α, CRP, and IL-6 levels and
also increases anti-inflammatory cytokines such as IL-10, IL-4, TGF-β (Plaisance et
al., 2006; Bruunsgaard et al., 2005; Hopps et al., 2011).
In the present study T1DM prolonged the time required for peak cell
contraction and the time to half relaxation of left ventricular cardiomyocytes, which
is in concert with recent studies conducted in our laboratory (Silva et al., 2011).
Cardiomyocytes of diabetic animals may reduce the expression of proteins such as
CaMKII, NCX, RyR2, SERCA2 and phospholamban (PLB). These changes lead to a
delay the availability of Ca2+ for cell contraction and affect the cardiac function of
diabetic rats (Bidasee et al. 2003; Bidasee et al. 2004; Bidasee et al. 2008, Stolen et
al. 2009, Choi et al., 2002). In addition, cardiomyocytes relaxation depends on the
removal of Ca2+ from the cytosol: to the sarcoplasmic reticulum (SR) by SERCA2
and PLB; to the extracellular medium by NCX and sarcolemmal Ca2+ ATPase; and to
the mitochondria by transport of mitochondrial Ca2+ (Bers, 2008). In diabetic
cardiomyopathy both expression and function of these cellular structures are reduced,
and hence the rate of Ca2+ removal from the cytosol is diminished (Rajesh et al.,
2012; Bidasse et al., 2008; Choi et al., 2002). Taken together, these changes
contributes to slow down cell relaxation (Loganathan et al., 2009).
In our study, left ventricular myocyte shortening was not altered by T1DM.
Previous studies show inconsistent results since unchanged (Howarth et al., 2002;
Howarth 2010) and reduced cardiomyocyte shortening (Silva et al., 2011; Choi et al.,
2002) are demonstrated in diabetic rats. Decreased sensitivity of contractile
myofilaments to Ca2+ and reduced intracellular concentration of Ca2+ are probable
mechanisms involved in the reduction cell shortening (Bers et al., 2008).
The swimming training employed here did not affect the contractile
properties assessed in the present study (cell shortening, time to peak and time to half
relaxation) in either control of diabetic rats. However, previous studies have shown
that exercise training can restore the contractile function of cardiomyocytes isolated
from diabetic animals (Loganathan et al. 2007; Shao et al. 2009; Stolen et al., 2009).
59
Adaptations to regular exercise can accelerate the availability of cytosolic Ca2+ and
increase the rate of ATP hydrolysis, increase the expression and/or the activity of
RyR2 and sensitivity of contractile myofilaments to Ca2+ in diabetic animals (Shao et
al., 2009). Moreover, the swimming program applied by Silva et al, (2011) reduced
the relaxation time of cardiomyocytes of diabetic animals. This effect has been
attributed to the ability of regular exercise to increase the speed of removal of Ca2+
from cytosol via increased expression of SERCA2 and PLB (Bers et al., 2008),
normalization of NCX expression and function, reduction in the phosphorylation of
CaMKII and restoration of the density of transverse tubules (Stolen et al., 2009).
Previous studies have demonstrated that cardiac myocyte unloaded shortening may
be affected or not by exercise training (Silva et al., 2011; Natali et al., 2002; Howarth
et al., 2010). It is worthy to note that, in vivo, cardiomyocytes are connected to each
other in an arrangement intimately associated to extracellular matrix and are subject
to varying mechanical loads along cardiac tissue. In fact, force development in
mechanically loaded cardiomyocytes from trained rats is significantly greater than in
those from sedentary rats (Natali et al., 2002). Probably some of the beneficial
effects of exercise on myocardial function may be due either to direct effects on
cardiac muscle and/or secondary effects including improved glucose metabolism
and/or alterations in collagen content of the heart and its associated effects on
compliance (Searls et al., 2004; Silva et al., 2011; Silva et al., 2013). Moreover, the
absence of effects of exercise training protocols on cardiomyocyte contractile
function may be due to insufficient intensity and/or duration of exercise.
In this study, exercise training had no significant effects on body mass in
either diabetic or control rats, a result that is consistent with previous studies
(Howarth et al., 2010; Silva et al., 2013; Silva et al., 2011). Pathological cardiac
hypertrophy induced by experimental diabetes has been related in previous studies.
In addition, the apparent absence of an effect of exercise on body mass in either
control or diabetic rats probably is due to anabolic and catabolic effects of the
exercise regime on muscle protein and fat metabolism, respectively.
Physical exercise is an important factor to improve the balance between
glycemic control, inflammation, and cardiovascular risk in T1DM (Rosa et al.,
2010). However, we observed that after a 12-hour overnight fast blood glucose was
not altered by exercise in either diabetic or control rats. These results are consistent
with other reports (Silva et al., 2011; Silva et al., 2013). It is possible that there has
60
been an increase in glucose uptake through GLUT1 and GLUT4 transporters via
activation of AMPK in ED animals and its counterregulatory action of glucagon has
helped in the maintenance of hyperglycemia (An and Robrigues, 2006). Studies have
demonstrated that exercise was able to improve glucose metabolism in diabetic rats
with the reduction of blood glucose levels (Aronson et al., 1997; Gomes et al., 2009).
However, it is not clear whether the apparent absence of an effect of exercise on
blood glucose level in diabetic rats may be due to the duration and intensity of
exercise protocol, the type and severity of diabetes in the animal model or the age of
the animals.
In this study T1DM reduced the resting HR in growing rats, but no statistical
difference between exercised and sedentary diabetic animals was found. Previous
studies reported a reduced HR in adult (De Angelis et al., 2000; Smirnova et al.,
2006), adolescent (Lucini et a., 2012) and growing rats with T1DM (Silva et al.,
2013), possibly due to autonomic dysfunction. In fact, bradycardia is a very early
indication of diabetic cardiomyopathy (Smirnova et al., 2006). Moreover,
cardiovascular autonomic neuropathy in diabetes commonly leads to abnormalities in
HR control and vascular dynamics (Maser at al., 2003). Additionally, degenerative
changes in autonomic neurons have been observed from three days to several weeks
after STZ injection in rats (De Angelis et al., 2000). On the other hand, exercise
training can prevent cardiac autonomic nervous dysfunction in diabetes (Chimen et
al., 2012; Yardley et al., 2012) and reverse bradycardia in these animals (De Angelis
et al., 2000).
Conclusion
In summary, our results demonstrate that left ventricle of growing rats with
STZ-induced diabetes displays decreased level of total and HMW adiponectin,
inflammatory response along with pathological structural remodeling. Low-intensity
swimming training promoted a non-significant increasing trend in the levels of IL10, attenuates TNF-α and pathological remodeling, and increases capillary density in
the left ventricle of these animals. These positive changes coexist with
cardiomyocyte contractile dysfunction and reduced HMW adiponectin. These results
provide insight into the beneficial effects of exercise on the myocardial
complications caused by T1DM.
61
Acknowledgements
The authors are grateful to the Fundação de Amparo à Pesquisa de Minas GeraisFAPEMIG (FAPEMIG) for the financial support (process CDS - APQ-01171-11/
PRONEX). AJ Natali is a CNPq fellow.
62
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CONCLUSÕES GERAIS
 Os efeitos crônicos do diabetes induzido por STZ ocorrem no miocárdio do
VE de ratos Wistar púberes e promovem cardiomiopatia diabética,
comprovada por alterações morfológicas e morfométricas.
 O VE de animais diabéticos apresentou
remodelamento estrutural com
aumento de colágeno total e de fibras reticulares na matriz extracelular,
acúmulo de glicogênio, desorganização da histoarquitetura do miocárdio,
infiltrado inflamatório e necrose de cardiomiócitos.
 Os níveis cardíacos de adiponectina total, adiponectina HMW, IL-10 e óxido
nítrico foram significativamente menores nos animais diabéticos.

O exercício físico não alterou de forma significativa os níveis cardíacos de
adiponectina total, adiponectina HMW e IL-10 no VE de ratos diabéticos e
não diabéticos.
 O diabetes ocasionou resposta inflamatória local com aumento dos níveis de
TNF-α no VE.
 O programa de natação de baixa intensidade atenuou a deposição de colágeno
total, o acúmulo de glicogênio, os níveis de TNF-α e as alterações
histopatológicas avaliadas no miocárdio do VE dos animais diabéticos.
 O percentual de capilares foi significativamente menor no VE dos animais
diabéticos.
 O programa de natação induziu aumento no percentual de capilares no
miocárdio dos animais diabéticos e não diabéticos e aumento nos níveis de
óxido nítrico no VE dos animais não diabéticos.
 Os cardiomiócitos isolados do VE dos animais diabéticos apresentaram
prolongamento do tempo para o pico de contração e do tempo para 50% de
relaxamento, mas não houve diferença na amplitude de contração.
 O programa de natação aplicado não alterou a amplitude de contração, o
tempo para o pico de contração e o tempo para 50% de relaxamento celular
em cardiomiócitos isolados do VE de ratos diabéticos e não diabéticos.
71